Methods of blocking cancer stem cell growth

ABSTRACT

Disclosed herein are antibodies against GPR49 and uses of such antibodies. The antibodies can be monoclonal, humanized, or fully human antibodies against GPR49, hybridomas or other cell lines expressing such antibodies, nucleic acids and vectors comprising nucleic acids encoding for such antibodies, and methods of blocking cancer stem cell growth with such antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of Application No.PCT/US2012/062862 entitled “METHODS OF BLOCKING CANCER STEM CELL GROWTH”filed Oct. 31, 2012 and published in English on May 10, 2013 as WO2013/067055 which claims the benefit of U.S. Provisional PatentApplication 61/554,426, filed on Nov. 1, 2011 and entitled METHODS OFBLOCKING CANCER STEM CELL GROWTH, the entirety of which is herebyincorporated by reference herein.

REFERENCE TO SEQUENCE LISTING

The present application is filed with a Sequence Listing in Electronicformat. The Sequence Listing is provided as a file entitledBIONO.001WO_Sequence.txt, created Oct. 25, 2012, which is approximately31 kb in size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD

This application relates generally to the field of cancer biology. Moreparticularly, embodiments are drawn to antibodies against GPR49 and usesof such antibodies. Several embodiments relate to monoclonal, humanized,or fully human antibodies against GPR49, hybridomas or other cell linesexpressing such antibodies, nucleic acids and vectors comprising nucleicacids encoding for such antibodies, and methods of blocking cancer stemcell growth with such antibodies.

BACKGROUND

G-Protein-coupled Receptor 49 (GPR49), also known as LGR5/HG38/FEX,belongs to the leucine-rich repeat containing G-protein-coupledreceptors (LGRs) that are structurally similar to glycoprotein hormonereceptors. LGRs are divided into three subgroups: (1) glycoproteinhormone receptors including thyroid-stimulating hormone (TSH) receptor,follicle-stimulating hormone (FSH) receptor, and luteinizing hormone(LH) receptor; (2) relaxin receptors LGR7 and LGR8; and (3) GPR48,GPR49, and LGR6. GPR49 is expressed in several tissues including theintestine, skeletal muscle, placenta, brain, and spinal cord. However,little is known about the function of GPR49.

SUMMARY

Several embodiments relate to a method of blocking cancer stem cellgrowth, comprising administering an effective amount of a monoclonalantibody that binds to G-Protein Coupled Receptor 49 (GPR49) polypeptidewith a K_(d) of less than 10×10⁻⁹ M to a cancer stem cell, wherein theGPR49 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 andthe effective amount is sufficient block cancer stem cell growth. In oneaspect, the cancer stem cell is a colon cancer stem cell. In anotheraspect, the cancer stem cell growth is measured as growth of a primarycolon cancer stem cell in serum-free and low-anchorage conditions invitro. In a further aspect, the effective amount of the antibody issufficient to reduce cancer stem cell growth by about 50%, about 40%,about 30%, or about 20% in comparison to cancer stem cells that have notbeen treated by the antibody.

In other aspects, the antibody is an IgG class antibody, an IgG1 classantibody, a human antibody, or a mouse antibody. In additional aspects,the GPR49 polypeptide consists of the amino acid sequence of SEQ ID NO:1.

In further aspects, the antibody is monoclonal antibody 14F7.1, 5F2.5,8E9.1, 76C12, 18G7.1, 78F05, 5D6.3, 1B3.5, 14A8.1, 5B10.1 or 7C3.4.

In various aspects, the antibody competitively inhibits a monoclonalantibody selected from the group consisting of 71C10, 86C11, 66D05,76C12, 78F05, and 76B04, or a monoclonal antibody produced by ahybridoma cell selected from the group consisting of monoclonal antibody2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5,6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4,2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1,4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1,18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2.

Certain embodiments are drawn to a method of producing an antibody orfragment thereof which specifically binds GPR49, comprising culturing ahost cell comprising a vector which comprises a polynucleotide sequenceencoding the antibody of claim 1; and recovering said antibody, orfragment thereof.

Another aspect is a monoclonal antibody that binds to G-Protein CoupledReceptor 49 (GPR49) polypeptide with a K_(d) of less than 10×10⁻⁹ M, forreducing growth-competent cancer stem cell abundance in a cellpopulation in a patient, wherein the GPR49 polypeptide comprises theamino acid sequence of SEQ ID NO: 1.

Yet another aspect is a monoclonal antibody that binds to G-ProteinCoupled Receptor 49 (GPR49) polypeptide with a Kd of less than 10×10-9M, for blocking cancer stem cell growth in a patient, wherein the GPR49polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing binding of anti-GPR49 Fabs to humanGPR49-Fc by ELISA.

FIG. 2 is a bar graph showing binding of anti-GPR49 Fabs to humanGPR49-HA transfected HEK293E cells by FACS.

FIG. 3 is a set of line graphs showing the EC50 values of 3 Dyax Fabcandidates on CHO-GPR49 cells (FIG. 3A) or parental CHO cells (FIG. 3B)by FACS binding.

FIG. 4 is a set of bar graphs showing binding of GPR49 Fabs to SW620colon tumor cells as measured by geometric mean (FIG. 4A) or percentpositive cells (FIG. 4B).

FIG. 5A is a bar graph showing binding of GPR49 Fabs to SW480 colontumor cells as measured by FACS geometric mean.

FIG. 5B is a bar graph showing binding of GPR49 Fabs to SW480 colontumor cells as measured by FACS percent positive cells.

FIG. 5C is a panel of FACS histograms of various concentrations of the76C12 Fab used to bind SW480 colon tumor cells.

FIG. 6A is a bar graph showing binding of GPR49 Fabs to HCT116 colontumor cells as measured by FACS geometric mean.

FIG. 6B is a bar graph showing binding of GPR49 Fabs to HCT116 colontumor cells as measured by FACS percent positive cells.

FIG. 6C is a panel of FACS histograms of various concentrations of the76C12 Fab used to bind HCT116 colon tumor cells.

FIGS. 7A-C are graphs showing binding of murine GPR49 antibodies to CHOGPR49-Flag-His expressing cells by FACs.

FIGS. 8A and 8C are graphs showing binding of murine GPR49 antibodies toCHO GPR49-Flag-His expressing cells by FACs. FIGS. 8B and 8D are tablesindicating the EC50 of the antibodies of FIGS. 8A and 8C, respectively.

FIGS. 9A, 9C, 9E, and 9G are graphs showing binding of second generationmouse antibodies to GPR49 by direct ELISA. FIGS. 9B, 9D, 9F, and 9H aretables indicating the EC50 of the antibodies of FIGS. 9A, 9C, 9E, and9G, respectively.

FIG. 10A is a bar graph showing the average number of tumorspheres fromsorted GPR49+, sorted GPR49−, or unsorted colon tumor cells. FIG. 10B isa microscopic image of GPR49+ and GPR49− cells in the tumorsphere assay.

FIG. 11 is a set of graphs showing tumor volume (FIG. 11A) and bodyweight (FIG. 11B) over time of GPR49+ MoFlo sorted colon tumor cellsinoculated into mice.

FIG. 12A is a picture of colonies formed by cells treated with the 76C12antibody (upper panel) or control IDEC152 treatment (lower panel) in thetumorsphere assay. FIG. 12B is a graph showing percent inhibition ofcancer stem cell tumorsphere growth of cells treated with the 78F05 or76C12 antibodies.

FIG. 13 is a graph showing inhibition of colon cancer stem cell sphereformation by treating cells with no antibody, isotype monoclonalantibody, or the 76C12 antibody.

FIG. 14 is a set of graphs showing Tumor Growth Inhibition for differentantibodies in a primary colon cancer model having K-Ras, PI3K, PTEN, andp53 mutations

FIG. 15 is a chart showing Percentage Tumor Growth Inhibition.

FIG. 16 is a chart showing Percentage Tumor Inhibition by GPR49mAb+Irinotecan combination treated CRC tumors with K-Ras, PI3K, PTEN,and p53 mutations.

FIG. 17 is a line graph showing Tumor Growth Inhibition in primary coloncancer model with K-Ras, PI3K, PTEN, H-Ras, APC, TP53, FGFR2, VANGL2,STK11, JAK2, and RB1 mutations.

FIG. 18A is a chart showing Anti-GPR49 antibodies reduce cancer stemcell frequency in colon cancer tumors with K-Ras, PI3K, PTEN, and p53mutations.

FIG. 18B is a graph showing that Anti-GPR49 antibody treatment inhibitsthe ability of CRC tumors antibodies with K-Ras, PI3K, PTEN, and p53mutations from re-forming new tumors when transplanted into secondaryrecipient mice.

FIG. 19 is a chart showing that GPR49 mAb 18G.7.1 treatment reduces thefrequency of cancer stem cells from CRC tumors with K-Ras, PI3K, PTEN,H-Ras, APC, TP53, FGFR2, VANGL2, STK11, JAK2, and RB1 mutations. CSCfrequency was measured in a serial re-implantation, limiting dilutionassay with primary CRC tumor cells previously treated as indicated.

FIG. 20 is a graph showing that Anti-GPR49 antibodies+Irinotecantreatment inhibits the ability of CRC tumors having K-Ras, PI3K, PTEN,H-Ras, APC, TP53, FGFR2, VANGL2, STK11, JAK2, and RB1 mutations to growupon re-implantation in secondary recipient mice.

DETAILED DESCRIPTION

Several embodiments of the present application are drawn to antibodiesagainst GPR49 and inhibiting cancer stem cell growth with suchantibodies. Various embodiments relate to humanized or fully humanantibodies against GPR49, hybridomas or other cell lines expressing suchantibodies, nucleic acids and vectors comprising nucleic acids encodingfor such antibodies, and methods of blocking cancer stem cell growthwith such antibodies.

Anti-GPR49 Antibodies

Several embodiments are drawn to anti-GPR49 antibodies. As used herein,GPR49 includes, but is not limited to, human GPR49 including thepolypeptide of NCBI Accession No. NP_003658.1 (SEQ ID NO: 1), which isencoded by the coding nucleotide sequence within NM_003667.2 (SEQ ID NO:2), or fragments thereof. The amino acid sequence of NCBI Accession No.NP_003658.1 and nucleotide sequence of NM_003667.2 are fullyincorporated by reference in their entireties. Examples of GPR49fragments contemplated herein include the GPR49 ectodomain,transmembrane domain, or intracellular domain and portions thereof.

As used herein, the term “antibody” includes, but is not limited to,synthetic antibodies, monoclonal antibodies, recombinantly producedantibodies, intrabodies, multispecific antibodies (including bi-specificantibodies), human antibodies, humanized antibodies, chimericantibodies, synthetic antibodies, single-chain Fvs (scFv), Fabfragments, F(ab′) fragments, disulfide-linked Fvs (sdFv) (includingbi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. The antibodies of severalembodiments provided herein may be monospecific, bispecific, trispecificor of greater multispecificity. Multispecific antibodies may be specificfor different epitopes of a polypeptide or may be specific for both apolypeptide as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tuft, etal., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992); each of which is incorporated herein by referencein its entirety.

Other anti-GPR49 antibodies contemplated include “oligoclonal”antibodies. As used herein, the term “oligoclonal” antibodies” refers toa predetermined mixture of distinct monoclonal antibodies. Methods forgenerating oligoclonal antibodies are known in the art. See, e.g.,“Examples Section”, example 1, PCT publication WO 95/20401; U.S. Pat.Nos. 5,789,208 and 6,335,163; each of which is incorporated herein byreference in its entirety. In certain embodiments, oligoclonalantibodies consist of a predetermined mixture of antibodies against oneor more epitopes generated in a single cell. In other embodiments,oligoclonal antibodies comprise a plurality of heavy chains capable ofpairing with a common light chain to generate antibodies with multiplespecificities (e.g., PCT publication WO 04/009618, which is incorporatedherein by reference in its entirety). Oligoclonal antibodies areparticularly useful when it is desired to target multiple epitopes on asingle target molecule (e.g., GPR49). Those skilled in the art will knowor can determine what type of antibody or mixture of antibodies isapplicable for an intended purpose and desired need. In particular,antibodies of several embodiments include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsto a GPR49 antigen (e.g., one or more complementarity determiningregions (CDRs) of an anti-GPR49 antibody). It is also specificallycontemplated that the antibodies of several embodiments includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds to a GPR49 antigen (e.g., one ormore complementarity determining regions (CDRs) of an anti-GPR49antibody).

Several embodiments are drawn to the anti-GPR49 human Fabs produced asdescribed in the Examples below, including antibodies 71C10, 86C11,66D05, 76C12, 78F05, and 76B04, which bind to a human GPR49-Fcectodomain (GPR49-RFc) (SEQ ID NO: 3). Several embodiments are drawn tothe full-length human IgGs of these Fabs as described in the Examples.

Several embodiments relate to the mouse monoclonal antibodies raisedagainst the human GPR49 ectodomain (GPR49-His) (SEQ ID NO: 4) asdescribed in the Examples below, including antibodies 2B5.5, 7F8.2,1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1,6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4,3G8.1, 5F2.5, and 6G10.1.

Several embodiments are drawn to the mouse monoclonal antibodies raisedagainst full-length human GPR49 as described in the Examples below,including antibodies 14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1,11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1,6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2.

The anti-GPR49 antibodies of several embodiments can be of any type(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Immunoglobulins may have both a heavy and light chain. An array of IgG,IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a lightchain of the kappa or lambda forms.

Several embodiments relate to a hybridoma that produces the light chainand/or the heavy chain of an anti-GPR49 antibody, including any one ofthe anti-GPR49 antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4,6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11,2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5,6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1,14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2,17C9.1, 2H5.1, and 10A9.2 produced and described in the Examples below.In one aspect, the hybridoma produces the light chain and/or the heavychain of a humanized or fully human monoclonal antibody.

Some embodiments are drawn to a nucleic acid molecule encoding the lightchain or the heavy chain of an anti-GPR49 antibody, including any one ofthe anti-GPR49 antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4,6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11,2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5,6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1,14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2,17C9.1, 2H5.1, and 10A9.2 produced and described in the Examples below.In one aspect, a nucleic acid molecule encodes the light chain or theheavy chain of a humanized or fully human monoclonal antibody.

Various embodiments are directed to a vector comprising a nucleic acidmolecule or molecules encoding a light chain and/or a heavy chain of ananti-GPR49 antibody, including any one of the anti-GPR49 antibodiesdesignated as 2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1,6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1,7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1, 14H9.1, 12G5.1, 6E10.1,14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2,1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2 producedand described in the Examples below.

Other embodiments relate to a method of making an anti-GPR49 antibodycomprising transfecting at least one host cell with at least one nucleicacid molecule encoding an anti-GPR49 antibody, expressing the nucleicacid molecule in said host cell and isolating said antibody. In severalaspects, such anti-GPR49 antibody includes any one of the anti-GPR49antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4, 10A6.7,10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5, 5G2.11,1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1, 14H9.1,12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1, 8E9.1,9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1, 2H5.1, and10A9.2 produced and described in the Examples below

In several embodiments, antibodies can specifically bind GPR49 andantigenic fragments thereof with a dissociation constant or K_(d)(k_(off)/k_(on)) of less than 10⁻⁵ M, or of less than 10⁻⁶ M, or of lessthan 10⁻⁷ M, or of less than 10⁻⁸ M, or of less than 10⁻⁹ M, or of lessthan 10⁻¹⁰ M, or of less than 10⁻¹¹ M, or of less than 10⁻¹² M, or ofless than 10⁻¹³ M.

In another embodiment, the antibody can bind to GPR49 and/or antigenicfragments thereof with a K_(off) of less than 1×10⁻³ s⁻¹. In otherembodiments, the antibody binds to GPR49 and antigenic fragments thereofwith a K_(off) of less than 10⁻³ s⁻¹, less than 5×10⁻³ s⁻¹, less than10⁻⁴ s¹, less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹,less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In another embodiment, the antibody binds to GPR49 and/or antigenicfragments thereof with an association rate constant or k_(on) rate of atleast 10⁻⁵ M⁻ s⁻¹, at least 5×10⁻⁵ M⁻¹ s⁻¹, at least 10⁻⁶ M⁻¹ s⁻¹, atleast 5×10⁻⁶ M⁻¹ s⁻¹, at least 10⁻⁷ M⁻¹ s⁻¹, at least 5×10⁻⁷ M⁻¹ s⁻¹, orat least 10⁻⁸ M⁻¹ s⁻¹, or at least 10⁻⁹ M⁻¹ s⁻¹.

An additional embodiment includes antibodies that have certain preferredbiochemical characteristics such as a particular isoelectric point (pI)or melting temperature (Tm).

In one embodiment, the high affinity antibodies have a pI ranging from5.5 to 9.5. In one embodiment, the high affinity antibodies of severalembodiments have a Tm ranging from about 65° C. to about 120° C.

Antibodies of several embodiments also encompass those that havehalf-lives (e.g., serum half-lives) in a mammal, (e.g., a human), ofgreater than 1 day, greater than 2 days, greater than 3 days, greaterthan 4 days, greater than 5 days, greater than 6 days, greater than 7days, greater than 8 days, greater than 9 days, greater than 10 days,greater than 15 days, greater than 20 days, greater than 25 days,greater than 30 days, greater than 35 days, greater than 40 days,greater than 45 days, greater than 2 months, greater than 3 months,greater than 4 months, or greater than 5 months. The increasedhalf-lives of the antibodies in a mammal, (e.g., a human), results in ahigher serum titer of said antibodies or antibody fragments in themammal, and thus, reduces the frequency of the administration of saidantibodies or antibody fragments and/or reduces the concentration ofsaid antibodies or antibody fragments to be administered. Antibodieshaving increased in vivo half-lives can be generated by techniques knownto those of skill in the art. For example, antibodies with increased invivo half-lives can be generated by modifying (e.g., substituting,deleting or adding) amino acid residues identified as involved in theinteraction between the Fc domain and the FcRn receptor (see, e.g.,International Publication Nos. WO 97/34631; WO 04/029207; U.S. Pat. No.6,737,056 and U.S. Patent Publication No. 2003/0190311 and discussed inmore detail below); each of which is incorporated herein by reference inits entirety.

In several embodiments, the antibodies may comprisemodifications/substitutions and/or novel amino acids within their Fcdomains such as, for example, those disclosed in Ghetie et al., 1997,Nat. Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund etal., 1991, J. Immunol. 147:2657-2662; Lund et al, 1992, Mol Immunol29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins etal., 1995, Proc Natl. Acad Sci USA 92:11980-11984; Jefferis et al, 1995,Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferiset al, 1996, Immunol Lett 54:101-104; Lund et al, 1996, Immunol157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogieet al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al,2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta etal., 2002, Biochem Soc Trans 30:487-490); U.S. Pat. Nos. 5,624,821;5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022;5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S.patent application Ser. No. 10/370,749 and PCT Publications WO 94/2935;WO 99/58572; WO 00/42072; WO 02/060919, WO 04/029207; each of which isincorporated herein by reference in its entirety. Othermodifications/substitutions of the Fc domain will be readily apparent toone skilled in the art.

Antibodies can comprise modifications/substitutions and/or novel aminoacid residues in their Fc regions that can be generated by numerousmethods well known to one skilled in the art. Non-limiting examplesinclude, isolating antibody coding regions (e.g., from hybridoma) andmaking one or more desired substitutions in the Fc region of theisolated antibody coding region. Alternatively, the variable regions ofan antibody may be subcloned into a vector encoding an Fc regioncomprising one or modifications/substitutions and/or novel amino acidresidues.

Antibodies of several embodiments may also be modified to alterglycosylation, again to alter one or more functional properties of theantibody.

In various embodiments, the glycosylation of the antibodies can bemodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for a target antigen.Such carbohydrate modifications can be accomplished by, for example,altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the antibody forantigen. Such an approach is described in further detail in U.S. Pat.Nos. 5,714,350 and 6,350,861; each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, an antibody of several embodiments can bemade that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies to thereby produce anantibody with altered glycosylation. See, for example, Shields, R. L. etal. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat.Biotech. 17:176-1, as well as, European Patent No: EP 1,176,195; PCTPublications WO 03/035835; WO 99/54342; each of which is incorporatedherein by reference in its entirety.

The antibodies of several embodiments can be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,antibodies can be recombinantly fused or conjugated to molecules usefulas labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387; each of which is incorporated herein byreference in its entirety.

Antibodies provided herein can include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingan GPR49 polypeptide or fragment thereof and/or generating a desiredresponse. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

In several embodiments, the antibodies specifically bind a polypeptidecomprising or consisting of a GPR49 polypeptide having at least 60%identity, or at least 70% identity, or at least 80% identity, at least85% identity, at least 90% identity, at least 95% identity, or at leastat least 97% identity, or at least 99% identity, or 100% identity to thehuman GPR49 polypeptide of NCBI Accession Nos. NP_003658.1 (SEQ IDNO: 1) or fragments thereof. Such fragments can, for example, be atleast about 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 contiguous ornon-contiguous amino acids of SEQ ID NO: 1, or any number of contiguousor non-contiguous amino acids in between any of the aforementionedlengths.

The percent identity of two amino acid sequences (or two nucleic acidsequences) can be determined, for example, by aligning the sequences foroptimal comparison purposes (e.g., gaps can be introduced in thesequence of a first sequence). The amino acids or nucleotides atcorresponding positions are then compared, and the percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=# of identicalpositions/total # of positions×100). The actual comparison of the twosequences can be accomplished by well-known methods, for example, usinga mathematical algorithm. A specific, non-limiting example of such amathematical algorithm is described in Karlin et al., Proc. Natl. Acad.Sci. USA, 90:5873-5877 (1993), which is incorporated herein by referencein its entirety. Such an algorithm is incorporated into the BLASTN andBLASTX programs (version 2.2) as described in Schaffer et al., NucleicAcids Res., 29:2994-3005 (2001), which is incorporated herein byreference in its entirety. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,BLASTN) can be used. See http://www.ncbi.nlm.nih.gov, as available onApr. 10, 2002. In one embodiment, the database searched is anon-redundant (NR) database, and parameters for sequence comparison canbe set at: no filters; Expect value of 10; Word Size of 3; the Matrix isBLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.

Several embodiments also encompass variants of the above describedantibodies, including any one of the anti-GPR49 antibodies designated as2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5,6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4,2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1,4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1,18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2 produced anddescribed in the Examples below, comprising one or more amino acidresidue substitutions in the variable light (V_(L)) domain and/orvariable heavy (V_(H)) domain. Several also encompass variants of theabove described antibodies with one or more additional amino acidresidue substitutions in one or more V_(L) CDRs and/or one or more V_(H)CDRs. The antibody generated by introducing substitutions in the V_(H)domain, V_(H) CDRs, V_(L) domain and/or V_(L) CDRs of the abovedescribed antibodies can be tested in vitro and in vivo, for example,for its ability to bind to GPR49 (by, e.g., immunoassays including, butnot limited to ELISAs and BIAcore).

In other embodiments, antibodies can have at least one, at least two, atleast three, at least four, at least five, or at least six of the CDRsof the antibodies described above, including any one of the anti-GPR49antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4, 10A6.7,10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5, 5G2.11,1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1, 14H9.1,12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1, 8E9.1,9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1, 2H5.1, and10A9.2 produced and described in the Examples below.

Various embodiments include antibodies that specifically bind to GPR49comprising derivatives of the V_(H) domains, V_(H) CDRs, V_(L) domains,or V_(L) CDRs of anti-GPR49 antibodies, such as any one of theanti-GPR49 antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4,10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5,5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1,14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1,8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1,2H5.1, and 10A9.2 produced and described in the Examples below, thatspecifically bind to GPR49. Standard techniques known to those of skillin the art can be used to introduce mutations (e.g., additions,deletions, and/or substitutions) in the nucleotide sequence encoding anantibody, including, for example, site-directed mutagenesis andPCR-mediated mutagenesis are routinely used to generate amino acidsubstitutions. In one embodiment, the V_(H) and/or V_(L) CDRsderivatives include less than 25 amino acid substitutions, less than 20amino acid substitutions, less than 15 amino acid substitutions, lessthan 10 amino acid substitutions, less than 5 amino acid substitutions,less than 4 amino acid substitutions, less than 3 amino acidsubstitutions, or less than 2 amino acid substitutions relative to theoriginal V_(H) and/or V_(L) CDRs. In another embodiment, the V_(H)and/or V_(L) CDRs derivatives have conservative amino acid substitutions(e.g. supra) made at one or more predicted non-essential amino acidresidues (i.e., amino acid residues which are not critical for theantibody to specifically bind to GPR49). Alternatively, mutations can beintroduced randomly along all or part of the V_(H) and/or V_(L) CDRcoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for biological activity to identify mutants thatretain activity. Following mutagenesis, the encoded antibody can beexpressed and the activity of the antibody can be determined.

Several embodiments also encompass antibodies that specifically bind toGPR49 or a fragment thereof, said antibodies comprising an amino acidsequence of a variable heavy chain and/or variable light chain that isat least 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical to the amino acid sequence of thevariable heavy chain and/or light chain of any of the antibodiesdescribed herein including any one of the anti-GPR49 antibodiesdesignated as 2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1,6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1,7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1, 14H9.1, 12G5.1, 6E10.1,14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2,1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2 producedand described in the Examples below.

Various embodiments further encompass antibodies that specifically bindto GPR49 or a fragment thereof, said antibodies or antibody fragmentscomprising an amino acid sequence of one or more CDRs that is at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99% identical to the amino acid sequence of one or moreCDRs of the antibodies described herein including any one of theanti-GPR49 antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4,10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5,5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1,14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1,8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1,2H5.1, and 10A9.2 produced and described in the Examples below. Thedetermination of percent identity of two amino acid sequences can bedetermined by any method known to one skilled in the art, includingBLAST protein searches.

Another embodiment includes the introduction of conservative amino acidsubstitutions in any portion of an anti-GPR49 antibody, such as any oneof the anti-GPR49 antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4,6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11,2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5,6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1,14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2,17C9.1, 2H5.1, and 10A9.2 produced and described in the Examples below.It is well known in the art that “conservative amino acid substitution”refers to amino acid substitutions that substitutefunctionally-equivalent amino acids. Conservative amino acid changesresult in silent changes in the amino acid sequence of the resultingpeptide. For example, one or more amino acids of a similar polarity actas functional equivalents and result in a silent alteration within theamino acid sequence of the peptide. Substitutions that are chargeneutral and which replace a residue with a smaller residue may also beconsidered “conservative substitutions” even if the residues are indifferent groups (e.g., replacement of phenylalanine with the smallerisoleucine). Families of amino acid residues having similar side chainshave been defined in the art. Several families of conservative aminoacid substitutions are shown in Table 1.

TABLE 1 Families of Conservative Amino Acid Substitutions Family AminoAcids non-polar Trp, Phe, Met, Leu, Ile, Val, Ala, Pro uncharged polarGly, Ser, Thr, Asn, Gln, Tyr, Cys acidic/negatively charged Asp, Glubasic/positively charged Arg, Lys, His Beta-branched Thr, Val, Ileresidues that influence Gly, Pro chain orientation aromatic Trp, Tyr,Phe, His

The term “conservative amino acid substitution” also refers to the useof amino acid analogs or variants.

Methods of Generating Antibodies

Antibodies that specifically bind to a GPR49 polypeptide can beidentified, for example, by immunoassays, BIAcore, or other techniquesknown to those of skill in the art.

The antibodies of several embodiments may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a GPR49 polypeptide can be administered to various host animalsincluding, but not limited to, rabbits, mice, rats, etc. to induce theproduction of sera containing polyclonal antibodies specific for theantigen. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and include but are not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981); each of which is incorporatedherein by reference in its entirety. The term “monoclonal antibody”(abbreviated as “mAb”) as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced. A “monoclonal antibody” may comprise, or alternativelyconsist of, two proteins, i.e., a heavy and a light chain.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with a GPR49 polypeptide ora cell expressing such peptide. Once an immune response is detected,e.g., antibodies specific for the antigen are detected in the mouseserum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a GPR49 polypeptide. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, several embodiments provide methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody wherein thehybridoma is generated by fusing splenocytes isolated from a mouseimmunized with a GPR49 antigen with myeloma cells and then screening thehybridomas resulting from the fusion for hybridoma clones that secretean antibody able to bind a GPR49 polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of severalembodiments may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

The antibodies of various embodiments can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular embodiment, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of several embodiments include those disclosed in PCTapplication No. PCT/GB91/01134; PCT publications WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which isincorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988), each of which is incorporated herein byreference in its entirety.

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988), each of which is incorporated herein by reference in itsentirety.

Human Antibodies and Humanization of Antibodies

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be desirable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397; each of which isincorporated herein by reference in its entirety. Humanized antibodiesare antibody molecules from non-human species antibody that bind thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and a framework region from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988); each of which isincorporated herein by reference in its entirety).

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089; each of which isincorporated herein by reference in its entirety), veneering orresurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)),de-immunization (U.S. Patent Publication No. 20030153043) and chainshuffling (U.S. Pat. No. 5,565,332), each of which is incorporatedherein by reference in its entirety.

Completely human antibodies can be used for therapeutic treatment ofhuman patients. Human antibodies can be made by a variety of methodsknown in the art including phage display methods described above usingantibody libraries derived from human immunoglobulin sequences. Seealso, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741, each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a GPR49 polypeptide. Monoclonal antibodies directed againstthe antigen can be obtained from the immunized, transgenic mice usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.13:65-93 (1995), which is incorporated herein by reference in itsentirety. For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., PCT publications WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; each of whichis incorporated herein by reference in its entirety. In addition,companies such as Abgenix, Inc. (Fremont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove. Abgenix, Inc. (Fremont, Calif.) provides XenoMouse® strains ofmice that have been engineered to contain up to but less than 1000kb-sized germline configured fragments of the human heavy chain locusand kappa light chain locus. See Mendez et al. Nature Genetics15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495(1998).

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, andusually a second constant region (preferably a gamma constant region)are formed into a construct for insertion into an animal. This approachis described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat.Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No. 0 546073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645,WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Human antibodies can be produced from transgenic mice that have hadlarge pieces of chromosomes or entire chromosomes introduced throughmicrocell fusion. See European Patent Application Nos. 773 288 and 843961, the disclosures of which are hereby incorporated by reference.Additionally, KM™-mice, which are the result of cross-breeding of Tcmice with Medarex's minilocus (Humab) mice have been generated. Thesemice possess the human IgH transchromosome of the Kirin mice and thekappa chain transgene of the Genpharm mice (Ishida et al., Cloning StemCells, (2002) 4:91-102).

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988), which is incorporated herein by reference in its entirety).

Further, antibodies to GPR49 polypeptides can, in turn, be utilized togenerate anti-idiotype antibodies that “mimic” GPR49 polypeptides usingtechniques well known to those skilled in the art (See, e.g., Greenspan& Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.147(8):2429-2438 (1991); each of which is incorporated herein byreference in its entirety).

In several embodiments, antibodies provided herein can be usedtherapeutically in vivo. Accordingly, the antibody can be modified tomake it less immunogenic in the individual. For example, if theindividual is human the antibody can be “humanized” where thecomplementarity determining region(s) of the antibody is transplantedinto a human antibody (for example, as described in Jones et al., Nature321:522-525, 1986; and Tempest et al., Biotechnology 9:266-273, 1991),which is incorporated herein by reference in its entirety.

Phage display technology can also be utilized to select antibody geneswith binding activities towards the polypeptide either from repertoiresof PCR amplified v-genes of lymphocytes from humans screened forpossessing anti-GPR49 antibodies or from naive libraries (McCafferty etal., Nature 348:552-554, 1990; and Marks, et al., Biotechnology10:779-783, 1992, which is incorporated herein by reference in itsentirety). The affinity of these antibodies can also be improved bychain shuffling (Clackson et al., Nature 352: 624-628, 1991, which isincorporated herein by reference in its entirety).

Methods of Producing Antibodies

The antibodies of several embodiments can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody or fragment, derivative or analogthereof, (e.g., a heavy or light chain of an antibody or a single chainantibody), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain) has been obtained, the vector for the production of the antibodymolecule may be produced by recombinant DNA technology using techniqueswell known in the art. Thus, methods for preparing a protein byexpressing a polynucleotide containing an antibody encoding nucleotidesequence are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Various embodiments thus provide replicablevectors comprising a nucleotide sequence encoding an antibody molecule,or a heavy or light chain thereof, or a heavy or light chain variabledomain, operably linked to a promoter. Such vectors may include thenucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464; each of which is incorporatedherein by reference in its entirety) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, several embodiments includehost cells containing a polynucleotide encoding an antibody, or a heavyor light chain thereof, or a single chain antibody, operably linked to aheterologous promoter. Vectors encoding both the heavy and light chainsmay be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules described herein. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule in situ. These include but arenot limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, NS0, 3T3, PerC6 cells) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).Bacterial cells such as Escherichia coli, and eukaryotic cells can beused for the expression of a recombinant antibody molecule. For example,mammalian cells such as Chinese hamster ovary cells (CHO), inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus are an effective expressionsystem for antibodies (Foecking et al., Gene 45:101 (1986); Cockett etal., Bio/Technology 8:2 (1990)). Also, see for example, U.S. Pat. Nos.5,827,739, 5,879,936, 5,981,216, and 5,658,759, each of which isincorporated herein by reference in its entirety.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), which is incorporated herein by reference in its entirety, inwhich the antibody coding sequence may be ligated individually into thevector in frame with the lacZ coding region so that a fusion protein isproduced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109(1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)), eachof which is incorporated herein by reference in its entirety; and thelike. pGEX vectors may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption and binding to matrix glutathione-agarose beads followedby elution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984), which isincorporated herein by reference in its entirety). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., Methods in Enzymol.153:51-544 (1987), which is incorporated herein by reference in itsentirety).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, NS0, Per.C6 and in particular, breast cancer cell linessuch as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normalmammary gland cell lines such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression can be used. For example, cell lines which stably express theantibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984),each of which is incorporated herein by reference in its entirety).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), each of which isincorporated herein by reference in its entirety.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987), which is incorporated herein by reference in its entirety). Whena marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983), whichis incorporated herein by reference in its entirety).

The host cell may be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors maycontain identical selectable markers which enable equal expression ofheavy and light chain polypeptides. Alternatively, a single vector maybe used which encodes, and is capable of expressing, both heavy andlight chain polypeptides. In such situations, the light chain should beplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, Nature 322:562 (1986); Kohler, Proc. Natl. Acad. Sci.USA 77:2197 (1980), which is incorporated herein by reference in itsentirety). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule has been produced by an animal, chemicallysynthesized, or recombinantly expressed, it may be purified by anymethod known in the art for purification of an immunoglobulin molecule,for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins. Inaddition, the antibodies or fragments thereof can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art, to facilitate purification.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In certainembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), which is incorporated herein by reference inits entirety, for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984), which is incorporated hereinby reference in its entirety) and the “flag” tag.

The antibodies described herein include derivatives that are modified(e.g., by the covalent attachment of any type of molecule to theantibody). For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching to said antibodies or antibody fragments polymermolecules such as high molecular weight polyethyleneglycol (PEG). PEGcan be attached to said antibodies or antibody fragments with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of said antibodies or antibody fragments orvia epsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

Further, antibodies can be conjugated to albumin in order to make theantibody or antibody fragment more stable in vivo or have a longer halflife in vivo. The techniques are well known in the art, see e.g.,International Publication Nos. WO 93/15199, WO 93/15200, and WO01/77137; and European Patent No. EP 413, 622, each of which isincorporated herein by reference in its entirety. Embodiments providedherein encompass the use of antibodies or fragments thereof conjugatedor fused to one or more moieties, including but not limited to,peptides, polypeptides, proteins, fusion proteins, nucleic acidmolecules, small molecules, mimetic agents, synthetic drugs, inorganicmolecules, and organic molecules.

Various embodiments encompass the use of antibodies or fragments thereofrecombinantly fused or chemically conjugated (including both covalentand non-covalent conjugations) to a heterologous protein or polypeptide(or fragment thereof, specifically to a polypeptide of at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90 or at least 100 amino acids) to generatefusion proteins. In some embodiments, the antibodies or fragmentsthereof can be recombinantly fused or chemically conjugated (includingboth covalent and non-covalent conjugations) to a heterologous proteinor polypeptide (or fragment thereof, specifically to a polypeptide of atleast about 10, at least about 20, at least about 30, at least about 40,at least about 50, at least about 60, at least about 70, at least about80, at least about 90 or at least about 100 amino acids) to generatefusion proteins. The fusion does not necessarily need to be direct, butmay occur through linker sequences. For example, antibodies may be usedto target heterologous polypeptides to particular cell types, either invitro or in vivo, by fusing or conjugating the antibodies to antibodiesspecific for particular cell surface receptors. Antibodies fused orconjugated to heterologous polypeptides may also be used in in vitroimmunoassays and purification methods using methods known in the art.See e.g., International publication No. WO 93/21232; European Patent No.EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat.No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al.,1991, J. Immunol. 146:2446-2452, each of which is incorporated herein byreference in its entirety.

Several embodiments include formulations comprising heterologousproteins, peptides or polypeptides fused or conjugated to antibodyfragments. For example, the heterologous polypeptides may be fused orconjugated to a Fab fragment, Fd fragment, FAT fragment, F(ab)2fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragmentthereof. Methods for fusing or conjugating polypeptides to antibodyportions are well known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EuropeanPatent Nos. EP 307,434 and EP 367,166; International publication Nos. WO96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci.USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; andVil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341, each ofwhich is incorporated herein by reference in its entirety.

Additional fusion proteins of antibodies that specifically bind GPR49 orfragments thereof (e.g., supra), may be generated through the techniquesof gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to alter the activities of antibodies orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2): 76-82; Hansson, et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313,each of which is incorporated herein by reference in its entirety.Antibodies or fragments thereof, or the encoded antibodies or fragmentsthereof, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. One or more portions of a polynucleotide encoding anantibody or antibody fragment, which portions specifically bind to aC/CLP may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In certainembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., 1989, Proc. Natl.Acad. Sci. USA 86:821-824, which is incorporated herein by reference inits entirety, for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the hemagglutinin “HA”tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., 1984, Cell 37:767, which isincorporated herein by reference in its entirety) and the “flag” tag.

Various embodiments further encompass antibodies or fragments thereofconjugated to a diagnostic or therapeutic agent. The antibodies can beused diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900, which isincorporated herein by reference in its entirety, for metal ions whichcan be conjugated to antibodies for use as diagnostics. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include but are notlimited to, ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc, in addition positron emittingmetals using various positron emission tomographies, nonradioactiveparamagnetic metal ions, and molecules that are radiolabelled orconjugated to specific radioisotopes can be conjugated to the antibodiesdescribed herein.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). A more extensive list oftherapeutic moieties can be found in PCT publication WO 03/075957, whichis incorporated herein by reference in its entirety.

The conjugates can be used for modifying a given biological response,the therapeutic agent or drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an apoptotic agent oran anti-angiogenic agent.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody in Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982), each of which is incorporated herein byreference in its entirety.

The antibodies can be conjugated to other polypeptides. Methods forfusing or conjugating antibodies to polypeptide moieties are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851, and 5,112,946; EP 307,434; EP 367,166; PCTPublications WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, PNASUSA 88:10535; Zheng et al., 1995, J Immunol 154:5590; and Vil et al.,1992, PNAS USA 89:11337, each of which is incorporated herein byreference in its entirety. The fusion of an antibody to a moiety doesnot necessarily need to be direct, but may occur through linkersequences. Such linker molecules are commonly known in the art anddescribed in Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson etal., 1999, Bioconjug Chem 10:553; Zimmerman et al., 1999, Nucl Med Biol26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171. Alternatively, anantibody can be conjugated to a second antibody to form an antibodyheteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, eachof which is incorporated herein by reference in its entirety.

Blocking Cancer Stem Cell Growth with Anti-GPR49 Antibodies

Several embodiments are drawn to blocking cancer stem cell growth withanti-GPR49 antibodies. In some embodiments, a method of blocking cancerstem cell growth comprises administering an effective amount of ananti-GPR49 antibody to cancer stem cells, wherein the effective amountof the anti-GPR49 antibody is sufficient to reduce growth of the cancerstem cells. In some aspects, an effective amount of an anti-GPR49antibody is administered to cancer stem cells in vitro. In otheraspects, an effective amount of an anti-GPR49 antibody is administeredto cancer stem cells in vivo.

As used herein, the term “cancer stem cell(s)” refers to a cell that canproliferate extensively or indefinitely and give rise to a largeproportion of cancer cells in a cancer. In some aspects, the largeproportion of cancer cells represents a majority of the cancer cells ina given cancer. For illustration, but not limitation, a cancer stemcell(s) can be a founder of a tumor or a progenitor of the cancer cellsthat comprise the majority of a cancer's mass.

Without being bound by theory, a cancer stem cell can have a variety ofcellular properties. For instance, a cancer stem cell can re-grow atumor as demonstrated by its ability to form tumors in animmuno-compromised mammal such as a mouse, and can form tumors uponsubsequent serial transplantation in immuno-compromised mammal such as amouse. Cancer stem cells can divide asymmetrically and symmetrically andcan show variable rates of proliferation. Additionally, a cancer stemcell can grow in vitro under established cancer stem cell conditionssuch as in a serum-free medium and/or in suspension or on low-attachmentplates.

Examples of cancer stem cells whose growth can be blocked by anti-GPR49antibodies include cancer stem cells from leukemias, such as acuteleukemia, acute lymphocytic leukemia, acute myelocytic leukemias, suchas, myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia leukemias and myelodysplastic syndrome (MDS); chronicleukemias, such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors such as but not limited to, glioma, astrocytoma, brain stemglioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungaling (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to papillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas.

As used with respect to blocking cancer stem cell growth, the term“effective amount” refers to an amount of anti-GPR49 antibody sufficientto reduce the growth of cancer stem cells by any degree. Any assay knownin the art can be used to measure cancer stem cell growth. For example,cancer stem cell growth can be measured by colony count, total cellcount, or volume/size of a cell population or colony. In severalembodiments, cancer stem cell growth can be measured by the tumorspheregrowth assay described below in the Examples.

In certain embodiments, an effective amount of an anti-GPR49 antibodycan block cancer stem cell growth as measured by at least a 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or100% reduction in the cancer stem cell population or tumorsphere growth,or any percentage in between any of the aforementioned numbers. In someaspects, the anti-GPR49 antibody is any one or combination of theanti-GPR49 antibodies designated as 2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4,10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5,5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1,14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1,8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1,2H5.1, and 10A9.2 produced and described in the Examples below.

For example, in some embodiments, an effective amount of an anti-GPR49antibody can block cancer stem cell growth as measured by at least about5%-99%, a 5%-80%, a 5 to 40%, a 10% to 99%, a 10 to 80%, a 10-60%, a10%-40%, a 20 to 99%, a 20%-80%, a 20%-60%, a 20%-40%, a 50%-98%,50%-80%, or a 60%-99% reduction in the cancer stem cell population ortumorsphere growth. In some aspects, the anti-GPR49 antibody is any oneor combination of the anti-GPR49 antibodies designated as 2B5.5, 7F8.2,1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1,6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4,3G8.1, 5F2.5, 6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1,11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1,6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2 produced and described in theExamples below.

In other embodiments, the effective amount of an anti-GPR49 antibody canblock cancer stem cell growth as measured by at least about a 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 10, 25, 50, 75, 100, 200, or1000-fold reduction in the cancer stem cell population or tumorspheregrowth, or any fold-reduction in between any of the aforementionednumbers. In some aspects, the anti-GPR49 antibody is any one orcombination of the anti-GPR49 antibodies designated as 2B5.5, 7F8.2,1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5, 6G10.3, 8H8.1,6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4, 2H9.2, 5B12.4,3G8.1, 5F2.5, 6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1, 4A10.2, 3F11.1,11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1, 18G7.1, 12E3.1,6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2 produced and described in theExamples below.

In some embodiments, the effective amount of an anti-GPR49 antibodysufficient to block cancer stem cell growth by any degree describedabove is in a concentration of about 1 nM, 50 nM, 75 nM, 100 nM, 150 nM,200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 500 nM, 550 nM, 600 nM, 700 nM,800 nM, 900 nM, 1 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300μM, 350 μM, 400 μM, 500 μM, 550 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM,75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM,900 mM, 1000 mM, 1 M, 5 M, 10 M, 15 M, 20 M, 25 M, 30 M, 35 M, 40 M, 45M, 50 M, 75 M, 100 M, or any number in between any two of theaforementioned concentrations. In some aspects, the anti-GPR49 antibodyis any one or combination of the anti-GPR49 antibodies designated as2B5.5, 7F8.2, 1B3.5, 9C6.4, 6H5.4, 10A6.7, 10A9.2, 2G8.1, 6C10.5,6G10.3, 8H8.1, 6B10.2, 3B8.11, 2F12.5, 5G2.11, 1F10.5, 10E1.1, 7C3.4,2H9.2, 5B12.4, 3G8.1, 5F2.5, 6G10.1, 14H9.1, 12G5.1, 6E10.1, 14F7.1,4A10.2, 3F11.1, 11F6.1, 5B10.1, 14A8.1, 8E9.1, 9C7.1, 4F6.2, 1B8.1,18G7.1, 12E3.1, 6H5.1, 2P69.2, 17C9.1, 2H5.1, and 10A9.2 produced anddescribed in the Examples below.

While the present embodiments have been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

EXAMPLES

Having generally described embodiments drawn to antibodies againstGPR49, hybridomas or other cell lines expressing such antibodies,nucleic acids and vectors comprising nucleic acids encoding for suchantibodies, and methods of blocking cancer stem cell growth with suchantibodies, a further understanding can be obtained by reference tocertain specific examples which are provided for purposes ofillustration only and are not intended to be limiting.

Example 1

Development of a Tumorsphere Assay to Maintain, Propagate and EnumerateColon Cancer Stem Cells from Primary Colon Tumors

An in vitro method was developed to maintain and propagate colon cancerstem cells based on the observation that normal and malignant stem cellsfrom neural and breast tissue can be maintained in non-adherentneurosphere or mammosphere cultures. These sphere cultures are grownunder “cancer stem cell” conditions: ultra-low-attachment plates toensure non-adherent cultures, with no serum and supplemented with growthfactors (i.e. EGF, bFGF) and anti-differentiation factors such asLeukemia-inhibitor-factor (LIF) (Singh et al., Nature 2004; Dontu etal., Genes Dev, 2003; Liu et al., Can Res 2006). Neuro- and mammosphereshave stem cell features such as the capacity for self-renewal anddifferentiation. Sphere cultures established from malignant tumortissue, termed tumorspheres, are highly tumorigenic in vivo, in additionto having self-renewal and differentiation capacity. Tumorspheres alsohave increased resistance to chemotherapeutic reagents, indicating thatthey are a relevant model for cancer drug development (Bao, 2006; Dean,2005; Diehn, Clarke 2006; Eyler, Rich 2008; Li 2008). Colon cancer stemcells can be maintained and propagated in vitro as primary colontumorspheres established from colon tumor biopsies (referred to asprimary colon tumors). Primary colon tumorspheres described belowsuccessfully established xenograft tumors in immune deficient with asfew as 10-100 cells. In contrast, typical standard tumor lines require1e6 or greater cells to successfully establish xenograft tumors.

Methods: The tumorsphere assay and cancer stem cells maintained in thetumorsphere cultures were used to design an assay to measure the numberof CSCs based on the hematopoietic bone marrow assay used to measuresstem/progenitor cells by their ability to form colonies inmethylcellulose (Bradley and Metcalf, 1966). In this assay, live singlecolon tumor cells derived from primary colon tumor xenografts or freshpatient biopsies were sorted by MoFlo and plated at between 1-1000 cellsper well into 96-well ultra-low attachment plates. The cells weremaintained in CSC medium for 3 weeks, with fresh medium exchangeperformed every 3-4 days. After 3 weeks, the number of cancer stem cellspresent in each well was determined by counting the number of coloniesor tumorspheres in each well using a GELCOUNT colony counter andsoftware (Oxford Optronix).

Results: Three tumorsphere models were developed using primary colonxenograft tumorspheres CT1 and CT3 (29), and the tumor line DLD-1 toassess anti-cancer stem cell (CSC) activity in vitro.

Example 2

Selection of Human Fabs Specific to Human GPR49 from Phage DisplayLibraries

Human antibodies that specifically recognize the extracellular domain ofa human GPR49 receptor were isolated using phage display technology.

Part I: Phage-Display Panning

Methods: Recombinant human GPR49-Fc ectodomain (GPR49-Fc) (SEQ ID NO: 3)was used to screen a human naive phagemid Fab library containing3.5×10¹⁰ unique clones (Hoet, R. M., et al. NatBiotechnol. 23(3): 344-8(2005)). Prior to incubation with the phage library, a biotinylatedanti-Fc antibody was captured on magnetic beads, followed by captured ofthe GPR49-Fc fusion protein. Selections were performed as described inHoet et al. After 3 rounds of panning, the 479 bp gene III stump wasremoved by Mlul digestion, and the vector was relegated for soluble Fabexpression in TGI cells.

Results: 61 unique clones were isolated in this panning. Unique cloneswere subsequently purified and binding was reconfirmed.

Part II: ELISA

Binding of Fabs to recombinant human GPR49-Fc ectodomain wasdemonstrated by ELISA. Methods: In brief, soluble GPR49-Fc fusionprotein at 2.5 ug/ml in 0.025 M carbonate buffer, pH 9.6 was coated at50 ul/well in a 96-well (IMMULON2 HB, Dynex Technologies, Inc., Cat.#3455) plate and incubated overnight at 4° C. The plate washed withphosphate-buffered saline (PBS, Irvine Scientific, Cat #9240), pH 7.4plus 0.025% Tween 20 in the Skan Washer 300 (Skatron Instruments),blocked with buffer containing 1% nonfat milk, 0.05% Tween 20 in PBS, pH7.4, and then incubated at room temperature for 1 hour. After incubationthe plate was washed with PBS plus 0.025% Tween 20 in the Skan Washer300. For the assay, the GPR49-coated plate was next incubated with thecontrol and test antibodies of varied concentrations, diluted in 1%nonfat milk, 0.05% Tween 20 in PBS at 50 ul/well. Following one hourincubation at room temperature, plate washed with PBS plus 0.025% Tween20 in the Skan Washer 300. A 2000-fold dilution in 1% nonfat milk, 0.05%Tween 20 in PBS of goat anti-human Kappa-HRP (Southern Biotech Cat#2060-05) was added 50 ul/well to detect bound Fab. Plate incubated for1 hour at room temperature washed with PBS plus 0.025% Tween 20 in theSkan Washer 300. TMB solution (KIRKEGAARD & PERRY LABS, INC. cat:50-76-00) was added 100 ul/well, and the reaction was stopped with 50ul/well of 4NH2S04 (LabChem, Cat # LC25830-0 after two minutes. Theabsorbance was measured at 450 nm, background 540 nm for TMB using theMolecular Devices plate reader. Data was analyzed using the SOFTMAX PROsoftware package version 4.3 LS (Molecular Devices Corp.) (FIG. 1).

Results: This resulted in six Fabs with titratable binding: 71C10,86C11, 66D05, 76C12, 78F05, and 76B04.

Part III: FACS Analysis

Methods: The six GPR49 Fabs were diluted 1:20, 1:40, and 1:80 and testedfor binding to HEK293E transfected with HA-GPR49 by FACS. Twenty-four toforty-eight hours post-transfection, cells are collected in suspensionand incubated on ice with anti-GPR49 antibodies or control IgG. Thecells were washed and primary antibodies detected with anti-mousesecondary antibodies conjugated to a fluorescent chromophore. Labeledcells were then sorted by FACS to identify anti-GPR49 antibodies thatspecifically recognize expression of native cell-surface GPR49 protein.

Results: All six prospective FACS positive GPR49 Fabs showed a decreasein binding to HA-GPR49-HEK293E with increasing Fab dilution and noneshowed binding to HEK293E (FIG. 2). Low geometric means may be due toFab epitopes not being very accessible, low expression of GPR49 on cellsurface, or Fabs may be low affinity. Additional testing by FACS ofGPR49 Fabs (diluted 2-fold from 400 nM down to zero) on CHO-GPR49 (50 nMMTX) compared to parental CHO revealed three Fabs with approximateEC50<10 nM: 76C12, 76B04, and 78F05 (FIGS. 3A and 3B). Testing of theFabs against tumor cell lines (SW480, SW620 and HCT116) revealed onlyFab 76C12 is capable of binding by FACS (FIGS. 4A, 4B, 5A-C, and 6A-C).

Part IV: Biacore Analysis

Methods: Three particular Fabs (76C12, 78F05 and 76B04) were identifiedthat specifically bound the human GPR49 receptor with less than 10 nMaffinity by FACS. To analyze the binding kinetics, biotinylatedanti-human IgG Fc antibody was immobilized on a Biacore SA chip to alevel of 2950 RUs. GPR49-Fc was then captured to a density of ˜400 RUsin flow cell 2 with flow cell 1 used as a reference. Purified Fabs (100,50, 25 nM in HSP-EP) were injected at 30 μl/min for 7 minutes with 20minutes allowed for dissociation. The data were analyzed withBIAevaluation software (v4.1) assuming a 1:1 model. To test the crossreactivity of the Fabs with the Fc domain, IgG1 Fc was then captured toa density of ˜250 RUs in flow cell 2 with flow cell 1 used as areference. All Fabs were then tested under the same conditions as above.All biacore experiments were performed at 25° C. on a BIAcore 3000instrument.

Results: Fabs 76C12 and 78F05 bound with K_(D)'s of 3.4 nM and 1.7 nMrespectively. Fab 76B04 exhibited weak binding.

Example 3

Construction of Full-Length Anti-GPR49 IgGs

Methods: Three Fabs were converted to human IgG1 and expressed in CHOcells. DNA sequences encoding three distinct anti-GPR49 Fabs 76C12,78F05 and 76B04 were selected from a human antibody phage library (DyaxCorp) by biopanning against a recombinant human GPR49 ectodomain-Fcfusion protein. The Fab gene sequences were used to construct expressionplasmids encoding full-length antibodies using the pV90AS expressionvector system for antibody production in mammalian cells. pV90AS is amodified pV90 expression vector designed to generate two transcriptsfrom a single promoter through alternate splicing of a primarytranscript (Reference: USPTO Application WO2005/089285). The natural CMVsplice donor is spliced either to a partially impaired splice acceptorto generate an antibody light chain-encoding transcript, or to a naturalCMV splice acceptor to generate the antibody heavy chain-codingtranscript. The partially impaired splice acceptor has been engineeredto result in similar amounts of both heavy and light chain transcripts.Light chain Variable (VL) and Constant (CL) regions were amplified byPCR. The 5′ light chain PCR primer included a Sfi I restrictionendonuclease site followed by sequence encoding an immunoglobulin lightchain signal peptide MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 5) in frame tosequences corresponding to the amino-terminus of the VL region accordingto the methods described in Nakamura T, et al., Int J Immunopharmacol.22:131-41 (2000), which is incorporated herein by reference in itsentirety. The PCR product was purified by agarose gel electrophoresisand extraction using the QIAquick GelExtration kit protocol (QIAGEN CA),digested with restriction endonucleases Sfi I and Asc I and ligated withthe Sfi I/Asc I digested pHLP025 vector (Holly Prentice). The pHLP025vector contains Sfi I/Asc I restriction endonuclease sites for receivingantibody light chain (signal peptide-VL-CL) as a Sfi I/Asc I digestedPCR fragment in addition to the natural CMV splice donor site sequence,a partially impaired splice acceptor site sequence, and a poly A signalsequence (Reference: USPTO Application WO2005/089285).

The heavy chain Variable (VH) region of each anti-GPR49 Fab (76C12,78F05, 76B04) was amplified by PCR. The 5′ heavy chain VH PCR included aNco I restriction endonuclease site followed by sequence encodingsynthetic heavy chain signal peptide MGWSLILLFLVAVATRVLS (SEQ ID NO: 5)in frame to sequences corresponding to the aminoterminus of the VHregion as described above. The 3′ heavy chain VH PCR primer includedsequence corresponding to the carboxyl-terminus of the VH region and anSfi I site. The PCR product was purified by agarose gel electrophoresisand extraction using the QIAquick GelExtration kit protocol (QIAGEN,CA), digested with restriction endonucleases Nco I and Sfi I and ligatedwith the Nco I/Sfi I digested pHLP029 vector (Holly Prentice). ThepHLP029 vector contains Nco I/Sfi I sites for receiving the antibodysignal peptide-VH sequence as a Nco I/Sfi I digested PCR fragment inaddition to an upstream poly A signal sequence, a natural CMV spliceacceptor site sequence, and a downstream poly A signal sequence(Reference: USPTO Application WO2005/089285).

The gene sequences coding for (Sfi I site-light chain signalpeptide-anti-GPR49 VL and CL) in pHLP025 and (heavy chain signalpeptide-anti-GPR49 VH-Sfi I site) in pHLP029 were assembled into asingle DNA fragment by PCR amplification through common overlappingsequences present in both vectors using the 5′ light chain and 3′ heavychain VH PCR primers described above. The resulting PCR product waspurified by agarose gel electrophoresis and extraction using theQIAquick GelExtration kit protocol (QIAGEN, CA), digested withrestriction endonuclease Sfi I and ligated with the Dra III digestedIgG1 parental vector.

Results: The resulting plasmid produces a bi-cistronic precursortranscript that upon alternative splicing results in translationallyactive antibody heavy and light chain mRNAs in approximatelystoichiometric quantities. Correct sequences were confirmed by DNAsequence analysis. Expression of full-length in mammalian cells resultedin production of stable, human IgG1 antibodies.

Example 4

Construction of Full-Length Anti-GPR49 IgGs for Improved Expression inMammalian Cells

To improve antibody expression yields and product quality the originalVH gene sequences from anti-GPR49 Fabs 76C12, 78F05, 76B04 weremodified.

Methods: First, anti-GPR49 VH sequences were analyzed for sequencescontaining putative splice sites with public sequence recognitionprograms (www.tigr.org/tdb/GeneSplicer/gene_spl.html (The Institute forGenomic Research, 9712 Medical Center Drive, Rockville, Md. 20850),www.fruitfly.org/seq_tools/splice.html). (Martin G. Reese and Frank H.Eeckman, Lawrence Berkeley National Laboratory, Genome InformaticsGroup, 1 Cyclotron Road, Berkeley, Calif., 94720; see also, Reese M G,Eeckman, F H, Kulp, D, Haussler, D, 1997. “Improved Splice SiteDetection in Genie”. J Comp Biol 4(3), 311-23). Second, codons in theheavy chain variable region of the anti-GPR49 Fabs were replaced withcodons corresponding to the identical Kabat positions from antibodiesthat have been successfully expressed in CHO cells without encounteringany changes in the original anti-GPR49 VH polypeptide sequence. Thissecond step mostly removes putative splice sites but an additionalsplice site analysis followed by synonymous codon exchange was performedto reduce the predicted likelihood of a putative splice site beingpresent.

DNA fragments encoding synthetic heavy chain leader in frame withsequence-optimized VH sequences of anti-GPR49 Fabs were obtained aschemically synthesized doublestranded DNA sequences from a commercialprovider (Blue Heron Biotechnology, Inc. Bothell Wash.). The Nco I andSfi I restriction endonuclease sites at 5′ and 3′ were included in thesynthesized fragments. The leader and anti-GPR49 sequence-optimized VHregion fragments were cloned into the Nco I/Sfi I digested the pHLP029vector as described in Example 3 above. Recombination with theappropriate corresponding light chains in pHLP025 and subsequent cloningof the single fragment is as described in Example 3 above. Correctsequences were confirmed by DNA sequence analysis.

Results: Expression of full-length antibodies from this plasmid seriesin mammalian cells results in increased production of stable, human IgG1antibodies.

Example 5

Transient Expression and Characterization of GPR49 Antibodies

Methods: Plasmid DNAs were used to transform CHO DG44 cells fortransient production of antibody protein. 20 u.g of plasmid DNA wascombined with 4×106 cells in a volume of 0.4 mL of 1×PBS. The mixturewas added to a 0.4 cm cuvette (BioRad) and placed on ice for 15 min. Thecells were electroporated at 600 uF and 350 volts with a Gene Pulserelectroporator (BioRad). The cells were placed into a T-25 flaskcontaining CHO-SSFM II media plus 100 uM Hypoxanthine and 16 uMThymidine and incubated at 37° for 4 days. In addition, plasmid DNA wasalso used to transfect 293E cells for transient expression of antibodyprotein. 1.2 u.g of each (heavy and light) plasmid DNA was transfectedinto 2×106 cells with Qiagen's Effectene Transfection Protocol (Qiagen,CA). Cells were incubated at 37° C. for 3 days.

Results: Supernatant was harvested and full-length antibody confirmed byboth Western Blot and ELISA methods. The ability of full IgG1 to bind toGPR49 was confirmed by ELISA.

Example 6

Development of Anti-GPR49 Antibody Producing CHO Cell Line

This example gives a detailed description of expression of theanti-GPR49 antibody comprising the binding domain of the Fab 76C12 asfull-length IgG1. The other Fabs described herein, i.e., those listed inExample 2, were expressed in a similar manner.

Methods: The variable and constant regions of 76C12 are of humansequence origin. The entire light chain and heavy chain variable regionsare derived from a Fab generated against human GPR49 by the DYAX phagedisplay technology. The variable, as well as the light chain constantregions were subcloned into an alternate splice expression vector. Thealternate splice configuration links the light and heavy chain throughthe usage of a single splice donor with two splice acceptors where eachsplice acceptor generates a transcript encoding one of the two chains.The expression vector DNA encoding the immunoglobulin genes waselectroporated into insulin independent Chinese hamster ovary cells (CHODG44i). A CHO transfectoma was selected for production purposes.

Complementary DNA from the corresponding variable (VL) and constant (CL)domains of the light chain gene of 76C12 and the variable (VH) domain ofthe heavy chain gene of 76C12 was cloned into an expression vector. Thevector contains cloning sites for inserting the entire light chain andvariable heavy cDNAs directly upstream of the human heavy chain constantregion. In addition to the Ig genes, this expression vector contains adihydrofolate reductase (DHFR) gene that can be used for selection inmammalian cells. The resulting expression vector was then transfectedinto CHO cells to initiate the generation of the anti-GPR49 secretingCHO cell lines.

The expression vector was electroporated into CHO cells. Immunoglobulinlight chain specific PCR primers were used to PCR amplify the Fab lightchain cDNA. The 5′ specific oligo sequence included the native signalpeptide from the light chain of the Biogen Idec anti-CD23 molecule. The5′ and 3′ oligos contain Sfi I and Asc I restriction endonucleaserecognition sequences, respectively, for subcloning into an intermediatevector. The VH cDNA was PCR amplified using a 5′ oligo that included asynthetic heavy chain signal peptide. The 5′ and 3′ oligos contain Nco Iand Sfi I restriction endonuclease recognition sequences, respectively,for subcloning into an intermediate vector.

Overlapping PCR using the light chain 5′ and VH 3′ oligos as templateswas employed to combine the light chain and the VH region as one cDNAsegment. The resultant product was subcloned into the Dra III site thuscreating the final alternate splice expression vector. The alternatesplice configuration generates two transcripts from a single promoterthrough alternate splicing of the primary transcript. The natural CMVsplice donor is spliced either to a suboptimal splice acceptor togenerate a light chain-encoding transcript, or to a natural CMV spliceacceptor to generate the heavy chain-coding transcript. The sub-optimalsplice acceptor has been designed to generate similar amounts of bothtranscripts.

The final DNA vector was prepared in HEBS buffer at a concentration of700 ng/uL prior to electroporation in to CHO cells. Fiveelectroporations were performed using various concentrations of DNA (15,20, 30, 40, and 45 ug). Each electroporation was done in a disposable0.4 cm cuvette (Invitrogen) containing 4×106 log phase CHO cells in 0.7ml sterile HEBS buffer and DNA in 0.1 mL HEBS (0.8 mL total volume).Cells were shocked using a Bio-Rad Gene Pulser XCELL, set at 290 volts,950 micro Faradays. Shocked cells were then allowed to stand at roomtemperature for 10 minutes then mixed with 10 mL room temp insulin freeCHOM16 medium, centrifuged (3′@ 1000 rpm), and aspirated. Cells werethen resuspended in 12 mL insulin free CHOM16 medium (RT) andtransferred to a T-75 tissue culture flask.

Cells and Media: prior to electroporation the CHO cells were grown inserum free media (CHOM24) with the addition of 1× nucleosides. CHOM24 isa chemically defined in-house media formulation that does not containany animal components. Methotrexate selection was performed innucleoside free CHOM16 and CHOM24 chemically defined media.

Following electroporation, 4×106 CHO cells were pooled into a T-75flask. Selection for DHFR expression began immediately as the cells wereinoculated in nucleoside free medium. Cells were eventually expanded to125 mL shake flasks in CHOM24 (˜3 weeks). To isolate clonal cell lines,the transfected stable pools were diluted and plated at 1 cell/well in200 uL CHOM16 on four 96-well plates. Plates were maintained at 37° C.until they were screened for antibody titer.

CHO colonies were screened for immunoglobulin production by assayingcell supernatants using an ELISA specific for the human kappa chain (day21 to day 28 after plating). The capture antibody used in the ELISA wasa polyclonal goat anti-human IgG (SouthernBiotech) and the detectionantibody was a polyclonal goat anti-human kappa conjugated tohorseradish peroxidase (SouthernBiotech). Colonies secreting the highestamount of immunoglobulin were expanded.

Results: High-expressing CHO cell lines were developed that resulted inanti-GPR49 mAbs with expected biochemical and biophysical propertiessuitable for scale-up and manufacturing.

Example 7

Purification and Characterization of Fully Human Anti-GPR49 IgG1Antibodies

The antibody produced in CHO cells were purified and characterized bymethods described below.

Methods: Protein A Capture: A Protein A column was pre-equilibrated with1×PBS (equilibration buffer) at 100-150 cm/hr with 3 column volumes.Supernatant was loaded at 150 cm/hr with a maximum of 10 mg of GPR49 mAbper milliliter of resin. After loading, the column was washed with 5column volumes of equilibration buffer. Then, the column was step elutedin an upflow direction with 100 mM Glycine, pH 3.0. Desired fractionswere collected and titrated to neutral pH with 2M Tris base. Collectedfractions were dialyzed against 1×PBS and concentrate material toprepare for the size exclusion step. A Size Exclusion aggregate removalstep involved equilibration of SUPERDEX 200 with 1×PBS with 1.5 columnvolumes at a flow rate of 36 cm/hr followed by loading of protein andcollecting desired fractions.

Identity testing was performed as follows:

1) Intact mass analysis by mass spectrometry where molecular massmeasurements were performed on an electrospray mass spectrometer(ESI-MSD). Prior to analysis, the sample was reduced to remove disulfidebonds. The deconvoluted mass spectrum represents the masses of the heavyand light chains.

2) N-terminal sequence analysis was performed by Edman degradation usingan ABI protein sequencer equipped with an on-line PTH analyzer. Thesequences for the initial amino acids of the light chain and heavy chainwere identified.

3) Peptide mapping with mass spectrometric analysis: tryptic or/andEndoLysC peptide maps were performed to obtain complete sequencecoverage by analysis of the LC/MS data generated from each peptide. Inaddition, determination of sites and amounts of oxidation anddeamidation were detected.

Purity testing was performed by; 1) SDS-Page or CE-SDS: Reduced andnon-reduced samples, this technique is used to measure antibodyfragmentation, aggregation and impurities, 2) SEC-HPLC with LS and RItechnique was used to measure aggregation and fragmentation and lightscattering determines the molar mass of sample components. 3) SDS gel orcapillary IEF method was used to determine the isoelectric focusingpattern and pi distribution of charge isoforms that can result from C-and N-terminal heterogeneity and/or deamidation. Finally, endotoxinconcentrations were measured by the Limulus amoebocyte lysate (LAL)kinetic turbidometric method.

Results: The purification of anti-GPR49 mAbs resulted in greater than99% monomer, endotoxin free mAbs in gram quantities with propertiessuitable for scale-up and manufacturing.

Example 8

Production of Mouse Antibodies to Human GPR49 Ectodomain

Part I: Hybridoma Selection

Methods: To produce antibodies against the ectodomain of GPR49, micewere immunized three times with purified and endotoxin free GPR49-His(SEQ ID NO: 4) using standard techniques. Blood from individual mice wasscreened for antigen recognition using ELISA and FACS analysis. Theanimals with the highest antibody titers were then selected for finalantigen boost after which spleen cells were isolated for hybridomaproduction. Approximately 1,000 clones were transferred from 4×24-wellfusion plates to 10×96-well culture plates. 199 positive clones wereselected by GPR49-Fc capture ELISA, and transferred to 48-well plates.From these, 100 positive clones were not selected because they eithershowed GPR49-CHO negative or positive binding to parental CHO by FACS.50 of the 99 positive clones were then selected depending upon isotype.

Results: 36 clones were sub-cloned (single or mixed IgGs bands, such as,IgG1/k, IgG2a/G and IgG1/2b/k). 8 clones lost expression from theparental to subclones. Monoclonal antibodies (mAbs) from 24 selectedsubclones were purified from the hybridoma supernatant using protein Aor protein G agarose chromatography and antibodies were tested by FACSas described below.

Part II: FACS Analysis

Methods: The 24 murine GPR49 mAbs were serially diluted and tested forbinding to CHO transfected with GPR49-Flag-His and parental CHO by FACS(standard methods).

Results: The results are summarized in Table 2 (data shown in FIGS.7A-C)

TABLE 2 GPR49 ms mAb FACs EC50 (nM) 2B5.5 ~0.2 7F8.2 ~0.2 1B3.5 0.81299C6.4 0.8297 6H5.4 ~1 10A6.7 1.428 10A9.2 4.522 2G8.1 9.766 6C10.5 9.9786G10.3 ~10 8H8.1 11.19 6B10.2 11.2 3B8.11 12.14 2F12.5 14.64 5G2.1120.94 1F10.5 24.54 10E1.1 29.49 7C3.4 34.51 2H9.2 43.27 5B12.4 43.563G8.1 63.83 5F2.5 530.5 6G10.1 2658Part III: Biacore Analysis

The binding kinetics of the mouse mAbs were analyzed by Biacore.

Methods: Biotinylated anti-human IgG Fc antibody was immobilized on aBiacore SA chip to a level of 2950 RUs. GPR49-Fc was then captured to adensity of ˜400 RUs in flow cell 2 with flow cell 1 used as a reference.Purified murine mAbs (100, 50, 25 nM in HSP-EP) were injected at 30μl/min for 7 minutes with 20 minutes allowed for dissociation. The datawere analyzed with BIAevaluation software (v4.1) assuming a 1:1 model.All biacore experiments were performed at 25° C. on a BIAcore 3000instrument.

Results: Murine mAbs bound to GPR49-Fc with 64 nM to less than 1 nMaffinities (K_(D)). Data are shown in Table 3.

TABLE 3 BIAcore mAb K_(D) nM 1B3-5 1.11 5B12.4 7.52 6C10.5 0.68 9C6.41.44 5G2.11 1.59 6G10_3 2.72 10E1_1 3.98 7F8.2 0.91 10A9.2 <100 pM6B10_2 3.05 2H9-2 0.71 2G8.1 N/A 6H5.4 0.17 10A6_7 <100 pM 3B8-11 3.92uM 3G8.1 0.99 7C3_4 6.32 2B5-5 1.67 8H8_1 1.54 2F12.5 1.29 1F10.5 64.05F2_5 5.52 6G10.1 0.14

Example 9

Production of Mouse Antibodies to Full-Length Human GPR49

Part I: Hybridoma Selection

Methods: To produce antibodies against the full length receptor, micewere immunized three times with 10 ug/mouse of DNA vector encoding thefull-length cDNA clone of the GPR49 gene mixed with gold particles.Blood from individual mice was screened approximately 75 days afterinitial immunization for antigen recognition using ELISA and FACSanalysis. The animals with the highest antibody titers were thenselected for final antigen boost (25 ug GPR49-Fc, 10 ug GPR49-DNA/goldparticle, and 5×10⁶ GPR49-CHO cells) after which spleen cells wereisolated for hybridoma production. Approximately 10,000 clones weregenerated with approximately 200 positive clones were selected byGPR49-CHO capture ELISA.

Results: 67 clones were confirmed by ELISA and GPR49-CHO FACS. 22 of thepositive clones were then sub-cloned (single or mixed IgGs bands, suchas, IgG1/k, IgG2a/G and IgG1/2b/k). Monoclonal antibodies (mAbs) from 19selected subclones were purified from the hybridoma supernatant usingprotein A or protein G agarose chromatography and antibodies were testedby FACS as described below.

Part II: FACS Analysis

Methods: The 19 murine GPR49 mAbs were serially diluted and tested forbinding to CHO transfected with GPR49-Flag-His and parental CHO by FACS(standard methods).

Results: Murine mAbs bound to GPR49 with 17 nM to less than 1 nM(EC50's). The results are summarized in Table 4 (data shown in FIGS.8A-D). Antibody 10A9.1 was used as a control.

TABLE 4 EC50 (nM, CHO- mAb GPR49) 14H9.1 0.9 12G5.1 1.2 6E10.1 1.314F7.1 1.5 4A10.2 1.7 3F11.1 1.8 11F6.1 1.9 5B10.1 1.9 14A8.1 2.1 8E9.12.2 9C7.1 2.2 4F6.2 2.2 1B8.1 2.2 18G7.1 2.5 12E3.1 2.6 6H5.1 3.0 2P69.25.1 17C9.1 12.5  2H5.1 17.5  10A9.2 (1st gen mAb) 6.2Part III: GPR49 Direct Binding ELISA

To characterize the binding of the mouse antibodies to GPR49, a directbinding ELISA assay was performed.

Methods: Soluble GPR49-Fc fusion protein at 2.5 ug/ml in 0.025 Mcarbonate buffer, pH 9.6 was coated at 50 ul/well in a 96-well (IMMULON2HB, Dynex Technologies, Inc., Cat. #3455) plate and incubated overnightat 4° C. The plate washed with phosphate-buffered saline (PBS, IrvineScientific, Cat #9240), pH 7.4 plus 0.025% Tween 20 in the Skan Washer300 (Skatron Instruments), blocked with buffer containing 1% nonfatmilk, 0.05% Tween 20 in PBS, pH 7.4, and then incubated at roomtemperature for 1 hour. After incubation plate was washed with PBS plus0.025% Tween 20 in the Skan Washer 300. For the assay, the GPR49-coatedplate was next incubated with the control and test antibodies of variedconcentrations, diluted in 1% nonfat milk, 0.05% Tween 20 in PBS at 50ul/well. Following one hour incubation at room temperature, plate washedwith PBS plus 0.025% Tween 20 in the Skan Washer 300. A 2000-folddilution in 1% nonfat milk, 0.05% Tween 20 in PBS of goatanti-mouse-Fc-HRP (Southern Biotech Cat #2060-05) was added 50 ul/wellto detect bound Fab. Plate incubated for 1 hour at room temperaturewashed with PBS plus 0.025% Tween 20 in the Skan Washer 300. TMBsolution (KIRKEGAARD & PERRY LABS, INC. cat: 50-76-00) was added 100ul/well, and the reaction was stopped with 50 ul/well of 4NH2S04(LabChem, Cat # LC25830-1) after two minutes. The absorbance wasmeasured at 450 nm, background 540 nm for TMB using the MolecularDevices plate reader. Data was analyzed using the SOFTMAX PRO softwarepackage version 4.3 LS (Molecular Devices Corp.) EC50 values wereobtained as a result.

Results: Murine mAbs bound to GPR49-Fc with EC50's in the range of 224nM to less than 1 nM (Table 5; FIGS. 9A-D).

TABLE 5 EC50 (ELISA) [nM] Hybridoma 0.2 8E9.1 0.24 11F6.1 0.25 12G5.10.27 5B10.1 0.28 6H5.1 0.38 14A8.1 0.5 9C7.1 0.53 4A10.2 0.55 14H9.10.55 14E3.1 0.57 14F7.1 0.65 4F6.2 0.657 6E10.1 0.67 2P69.2 0.88 3F11.10.95 17C9.1 1.34 1B8.1 1.35 18G7.1 2.86 2H5.1 225.4 18D9.1

Example 10

Cancer Tumors can be Sorted with Anti-GPR49 Antibodies

Methods: The expression of GPR49 in colon and other tumor cells wasmeasured by flow cytometry (FACs) using the anti-GPR49 mAbs 76C12 and78F05.

Results: GPR49 was found to be expressed in multiple colon tumors (CT1,CT3, LS174T, Sw480, HCT116, SW620, DLD-1, Lovo), gastric tumor N87, lungtumor A549, and the positive control GPR49-CHO stable transfectants. Inaddition, the specificity of anti-GPR49 mAb binding to GPR49 wasconfirmed by RNAi knockdown of GPR49 in LS174T cells, whichsignificantly inhibited the binding of GPR49 mAbs 76C12, 76B04, 78F05 tobind LS174T cells.

Example 11

Validation of GPR49 as a Marker of Colon Cancer Stem Cells (CSC)

The colon tumorsphere assay described in Example 1 was used to determinewhether colon tumor cells isolated based on anti-GPR49 mAb binding wereenriched for cancer stem cells.

Part I:

Methods: CT1 primary colon tumor cells maintained in cancer stem celltumorsphere conditions were sorted by FACs using GPR49 mAbs 76C12. GPR49positive (GPR49+), GPR49 negative (GPR49−) and unsorted live PI(propidium iodide) negative cells were seeded at 1 cell per well in 96well plates, and analyzed 3 weeks later for sphere forming ability (i.e.number of cancer stem cells).

Results: The sorted GPR49 positive (GPR49+) colon tumor cells werehighly enriched for cancer stem cell activity compared to GPR49 negativeand unsorted cells (FIGS. 10A and 10B). GPR49 sorted LS174T colon tumorcells were similarly highly enriched for cancer stem cells in 2separate, independent experiments.

Part II:

Methods: To further corrobate these results, we performed an additionalexperiment where colon cancer stem cell tumorpheres were treated withGPR49 or control RNAi-1 and then seeded in 96-well plates at 250 cellsper well.

Results: Treatment with GPR49 RNAi-1 but not control RNAi significantlyreduced the number of colon cancer stem cells measured 14 days aftertreatment.

Example 12

GPR49 Positive Colon Tumor Cells have Cancer Stem Cell Properties InVivo

Methods: To show that isolated GPR49+ cells have cancer stem cellproperties, in vivo cancer stem cell assays comparing the in vivo growthof GPR49+ versus GPR49− cells were performed. GPR49+ cells from theprimary colon tumor line CT1 were sorted with 76C12 using a MoFlo, andimplanted at 1000 cells per mouse. Mice were analyzed for tumorformation and weight loss over the next 50 days.

Results: GPR49+ primary colon tumor cells cause aggressive tumor growth(FIG. 11A) and rapid weight loss (FIG. 11B) in a primary tumor xenograftstudy. In contrast, GPR49− cells grew very slowly and did not causerapid weight loss. These findings demonstrate that GPR49 high expressingcolon tumor cells are highly tumorigenic, an important feature of cancerstem cells.

Example 13

R-Spondins Bind to GPR49 with High Affinity but do not ActivateGPR49-Mediated Signalling

Part I:

GPR49 is an orphan G protein-coupled receptor (GPCR) related to theglycoprotein hormone receptor family (eg. receptors for FSH, TSH, LH)and is an intestinal and stomach stem cell marker that was identified byscreening Wnt target genes in the gut (Barker et al., Nature, 2007). Toidentify the natural ligand of GPR49, binding studies were focused onknown Wnt-pathway modulators Noggin, Gremlin1, DAN, Chordin-like 1,Cerberus1, PRDC, stanniocalcin-1, COCO, Chordin, R-spondin1-3, BMP2 andBMP4.

Methods: Ligand binding activity to GPR49-Fc was determined in abiolayer interometry-based assay. In brief, GPR49-Fc and test ligands(all purchased from R& D systems) were all diluted in OB buffer (PBS, pH7.4, 0.01% (w/v) NaN₃, 1 mg/ml BSA, 0.02% (v/v) Tween 20). GPR49-Fc wascaptured on anti-human IgG Octet tips (ForteBio, Inc., Menlo Park,Calif.; Part #18-5001), using an Octet Red System (ForteBio, Inc., MenloPark, Calif.). Tips were washed in OB buffer and moved to wellscontaining test ligand in OB buffer. Binding of test ligand to GPR49-Fcwas recorded as biointerferometry signals during an association phase(120 seconds) and dissociation phase (120 seconds). In addition, theligands were tested against murine GPR49-Fc.

Results: R-spondin (RSPO) family members interact to human GPR49-Fc, butshow a non-specific component that makes interpretation difficult.Specifically, the two-phase association and dissociation observedsuggests multiple binding events. RSPO family members do not interactwith mouse GPR49-Fc [88% identity (91% similar)].

Part II: Although the GPR49-Fc used is a dimer of GPR49 ecto-domain insolution (due to interacting Fc halves), the bi-phasic binding cannot beexplained by this due to the immobilization of the GPR49-Fc to the tipand the monomeric state of the test RSPO molecules. Therefore, thebi-phasic nature of the RPOS binding to GPR49-Fc was furtherinvestigated by a solution affinity surface plasmon resonance assay.

Methods: Binding of RSPO to GPR49 was analyzed using solution affinitysurface plasmon resonance (Day E S, et al. Biochemistry. 2005 Feb. 15;44(6):1919-31). The method utilizes conditions of so-called“mass-transport-limited” binding, in which the initial rate of ligandbinding (protein binding to the sensor chip) is proportional to theconcentration of ligand in solution (BIApplications Handbook (1994)Chapter 6: Concentration measurement, pp 6-1-6-10, Pharmacia BiosensorAB). Under these conditions, binding of the soluble analyte (proteinflowing over chip surface) to the immobilized protein on the chip isfast compared to the diffusion of the analyte into the dextran matrix onthe chip surface. Therefore, the diffusion properties of the analyte andthe concentration of analyte in solution flowing over the chip surfacedetermine the rate at which analyte binds to the chip. In thisexperiment, the concentration of free RSPO-1 in solution is determinedby the initial rate of binding to a CM5 Biacore chip containingimmobilized GPR49-Fc. Into these RSPO-1 solutions were titrated thecompeting GPR49-Fc.

Results: Initial binding rates were obtained from raw sensorgram data.R-spondin-1 showed a bi-phasic binding profile suggesting multiplecooperative binding sites for RSPO on GPR49.

Part III. To test for the effects of RSPO on GPR49 signaling, multipleassays were utilized.

Methods: Cyclic AMP, Calcium flux and β-arrestin assays were conductedby standard methods. In addition, a β-catenin/TCF reporter assay whichis a transcription-based reporter assay that characterizes theWnt/beta-catenin signaling pathway. The β-catenin/TCF reporter assayutilized transfected cells with SAbioscience TCF/LEF reporter ornegative construct. After 24 hour transfection, cells were counted andaliquoted (2×10⁴ cells/well) into a 96 well plate. Cells were thenstarved with Opti-MEM+0.5% FBS+0.1 mM NEAA+1 mM sodium pyruvate+1×antibiotic media for 6 hours. Cells were then treated with titratedamount of RSPO and mWnt3a (200 ng/ml) with or without antibody for 18hours. Dual luciferase activities are developed with Promega dualluciferase kit. In addition, GPR49 RNAi was used to examine the specificcontribution of GPR49 to RSPO dependent β-catenin/TCF reporter activity.

Results: No measurable activity was observed in the cAMP, calcium fluxor β-arrestin activity assays for RSPO. However, RSPO droveβ-catenin/TCF reporter activity in a dose-dependent manner that wasknocked down by GPR49 RNAi.

Example 14

Stimulation of GPR49-3T3 Cells with R-Spondin-1

Methods: To test the effects of R-spondin on the proliferation of GPR49overexpressed cells, 2500 NIH 3T3 fibroblast cells stably transfectedwith GPR49 (GPR49-3T3 clone 50) were incubated with R-spondin 1 for 2days and the Cell Titer Glo ATP assay used to assess cell proliferation.GPR49-3T3 clone 50 was previously shown to express high levels of cellsurface GPR49 by FACs with anti-GPR49 mAb 76C12.

Results: R-spondin 1 stimulation caused a 25-40% increase in cellproliferation of GPR49-3T3 cells compared to control stimulation withStanniocalcin in serum free conditions. This result was confirmed in asecond independent experiment using a different clone, GPR49-3T3 clone28, which showed a 4-fold increase in proliferation in response toR-spondin 1 compared to Stanniocalcin control stimulated cells.

Example 15

Inhibition of R-Spondin Binding by GPR49 mAbs

The ability of the anti-GPR49 mAbs detailed in this invention to blockRSPO binding to soluble GPR49-Fc was determined.

Methods: The ligand blocking ability of the anti-GPR49 antibodies wasdetermined by a solution phase competition surface plasmon resonanceassay. Briefly, the antibodies (1 uM) co-incubated with 200 nM RSPO for45 minutes on ice. RSPO alone (200 nM) or in combination with incubatedantibodies were then flowed over a CM5 Biacore chip immobilized withGPR49-Fc (as detailed in Example 2). The R max, which is the bindingsignal at the end of the association phase of 60 seconds (R max) wasused as a measure of the fraction bound at stead state/equilibrium.

Results: Anti-GPR49 antibodies 3B8.11, 10A6.7, 2B5.5, 6C10.5 alldecreased R max great than 20%. 3G8.1, 6H5.4 and 7F8.2 all decreased theR max by less than 20%.

Example 16

GPR49 mAbs Cross-Reactivity and Specificity to Related Family MembersGPR48 (LGR4) and LGR6

Methods: In order to determine the specificity of the anti-GPR49antibodies detailed in this invention for related family members, GPR48(LGR4) and LGR6, recombinant GPR48, GPR49 and LGR6 were independentlyexpressed in mammalian cells (HEK293T). Binding of antibodies to cellsoverexpressing the receptors was assessed by FACS (standard methods) andcompared to cells transfected with control vector (pV100).

Results: 76C12 antibody binds to GPR48 but not to LGR6 expressed inmammalian cells. None of the other antibodies test bind to either GPR48or LGR6 (Table 6)

TABLE 6 Human GPR48 Human (LGR4) LGR6 76C12 Y N 78F05 N N 1B8.1 N N14E3.1 N N 14A8.1 N N 14F7.1 N N 18G7.1 N N 6H5.4 N N 7C3.4 N N 7F8.2 NN 14H9.1 N N 9C7.3 N N 1B3.5 N N

Example 17

Internalization of GPR49 by Fully Human Anti-GPR49 Antibodies

Methods: Lovo colon tumor cells were seeded at 50,000 cells per wellinto 8 well chamber slides (Becton Dickinson Collagen Type 1 coatedculture slides, BD BioCoatTM #354630) 48 hours prior to stainingprocedures. Cells were routinely maintained below 20 passages. On day ofstaining procedures, culture media was discarded from each well andreplaced with 500 ul cold incubation buffer (MEM Eagle ATCC #30-2003+1%BSA). Cells were washed 2× with this buffer for 3 min each wash. 250 ulof each mAb (76C12, 78F05 and murine 10A9.2) to be tested was then addedto the appropriate well at a concentration of 10 ug/ml, diluted inincubation media, and incubated on ice for 1 hour. A humananti-human-IGF-1R antibody was used as a positive control to comparedegree of internalization. Antibody 5A7 (anti-Id), IDEC152 and noantibody were used as negative controls. After the 45 minutes incubationon ice, the time zero (t=0′) slide washed 3× with 500 ul of cold washbuffer (PBS+1% BSA+2% Goat serum) for 3 min each wash (slides alwayskept on ice). The t=0 slide was then fixed with 500 ul 14%paraformaldehyde (diluted with PBS from 16% stock; EMS #15710) for 15minutes at room temperature. The t=0 slide was then washed again 3× withcold wash buffer for 3 minutes each wash, then left on ice. Meanwhile,the remaining slides were put into a 37° C. incubator for theirdesignated time points (15 and 60 minutes). At the end of theirincubation time each slide followed the same procedures as above—washesand fixation, and put on ice. All slides were then permeabilized with200 ul cold permeabilization buffer (Wash buffer+0.5% Triton-X) for 10minutes on ice. All slides were then washed 3× with 500 (×1 cold washbuffer for 3 minutes each wash. The secondary antibody was prepared at a1:1000 dilution (AlexaFluor 488 Goat-anti-mouse IgG (H+L), MolecularProbes # A11029 for the mAbs and AlexaFluor 488 Goat-anti-human IgG(H+L), Molecular Probes # A11013 for G4 antibodies) in wash buffer,after an initial spin of the stock vial at 10,000 rpm for 10 min at 4°C. 250 ul of the diluted secondary antibody was added to each well andincubated for 40 min at room temperature in the dark (covered). Slideswere again washed 3× with 500 ul cold wash buffer. On the final wash,the buffer was discarded and all wells were left empty. The chamberswere then disassembled from the slide using the provided disassemblytool, and cover slips were mounted with Vectashield mounting mediumcontaining DAPI (Vector # H-1500, Hard Set™). Slides were stored at 4°C. in the dark overnight to allow the mounting medium to dry. Picturesof the slides were taken with a confocal microscope using the LaserSharp2000 program (BioRad v5.2) and represented as a merge of blue and greencomponents from Kalman 10 average.

Results: 76C12, 78F05 and 10A9.2 all showed rapid internalization ofGPR49 in 60 min. As expected the positive control, IGF1R C06, showedinternalized of the IGF1R receptor whereas isotype matched negativecontrols (mouse 5A7, IDEC152 (primatized antibody)) did not bind orinternalize.

Example 18

GPR49 Antibodies Bind to Murine GPR49

Methods: In order to determine the specificity of the anti-GPR49antibodies described herein to murine GPR49, recombinant murine GPR49was expressed in mammalian cells (HEK293T). Binding of antibodies tocells overexpressing the receptors was assessed by FACS (standardmethods) and compared to cells transfected with control vector (pV100).

Results: 76C12 binds to murine GPR49 expressed in mammalian cells withhigh affinity. Multiple additional GPR49 mAbs bind to murine GPR49(Table 7).

TABLE 7 murine GPR49 (Lgr5) 76C12 HI 78F05 LO 1B8.1 MED 14E3.1 MED14A8.1 MED 14F7.1 MED 18G7.1 LO 6H5.4 LO 7C3.4 LO 7F8.2 LO 14H9.1 LO9C7.3 +/− 1B3.5 +/−

Example 19

Epitope Grouping of Anti-GPR49 mAbs

An equilibrium binding assay was developed and used to determine commonepitope binding groups for the panel of GPR49 antibodies detailed inthis invention. Cross-blocking of binding to GPR49 was used to definegroups of antibodies that bind to distinct binding epitopes of GPR49.

Methods: GPR49-Fc binding activity to the test anti-GPR49 mAb wascompared to that of GPR49-Fc pre-incubated with secondary test mAb(either self or different mAb) in a biolayer interometry-based assay.Primary anti-GPR49 test mAb (primary mAb) was biotinylated with a ThermoScientific EZ-Link Sulfo-NHS-Biotin (Thermo Scientific, #21425)according to manufacturer's protocol. Biotinylated primary mAb,GPR49-Fc, and GPR49-Fc plus secondary test anti-GPR49 mAb (secondarymAb) were all diluted in OB buffer (PBS, pH 7.4, 0.01% (w/v) NaN3, 1mg/ml BSA, 0.02% (v/v) Tween 20). Biotinylated primary mAbs werecaptured on streptavidin Octet tips (ForteBio, Inc., Menlo Park, Calif.;Part #18-5001), using an Octet Red System (ForteBio, Inc., Menlo Park,Calif.). Tips were washed in OB buffer and moved to wells containingGPR49-Fc in OB buffer. Binding of GPR49-Fc to the primary mAb on thetips was recorded as biointerferometry signals to saturation, during anassociation phase (120 seconds) and the binding signal at the end of theassociation phase (R max) was used as a measure of the fraction bound atstead state/equilibrium. Binding during the dissociation phase (120seconds) was also recorded. To measure the cross-blocking ability of thevarious mAbs to each other, a second binding experiment was performed inwhich the GPR49-Fc outlined above, was preincubated with a 5 fold molarexcess of secondary test mAb (unbiotinylated). This was then bound tothe tips pre-load with biotinylated primary mAb to determine the R max′.The R max′ prime was then compared to the R max and the percentage of Rmax′/R max was calculated to determine the amount of blocking thesecondary mAb had on the ability of GPR49-Fc to bind to the primary mAbpre-bound to the tip. Cross-blocking (R max′/R max×100) of secondary toprimary mAbs was recorded as follows: 0-25% full cross-blocking, 25-50%partial cross-blocking, 50-75% low cross blocking, 75-100% nocross-blocking. All mAbs were tested against self (primary and secondarymAb were the same test mAb) to ensure proper analysis.

Results: Six distinct epitope binding groups were observed for theanti-GPR49 mAbs tested. The groups are: group 1 (76C12, 1B3.5, 6B10.2,4F6.2), group 2 (18G7.1, 14A8.1, 5B10.1, 14F7.1, 11F6.1, 14E3.1, 1B8.1),group 3 (5F2.5, 6B10.2), group 4 (3F11.1), group 5 (10A.2) and group 6(6E10.1). Cross-blocking by self-mAbs is shown in Table 8 and 9.

TABLE 8 18G7.1 1B3.5 76C12 14A8.1 5B10.1 14F7.1 5D6.3 5F2.5 18G7.1  11100 100  20  28  14  62 100 1B3.5 100  4  7 100 100 100  47 100 76C12100  4  5 100 100 100  25 100 14A8.1  5 100 100  6  9  4  99 100 5B10.1 13 100 100  16  11  14  76 100 14F7.1  7 100 100  7  11  5 100 1005D6.3 100  96  81 100 100 100  66  97 5F2.5 100 100 100 100 100 100  79 13 11F6.1  9 100 100  93 3F11.1  78  68 100 100 14E3.1  5  88  95  831B8.1  6  85  96  85

TABLE 9 11F6.1 3F11.1 14E3.1 1B8.1 10A9.2 6B10.2 4F6.2 6E10.1 18G7.11B3.5 76C12 100  4  6 100 14A8.1 5B10.1 14F7.1 5D6.3 5F2.5 100  8 100100 11F6.1  4 99  0  1 100 100 100 100 3F11.1 100  5 86 75 100 100 100100 14E3.1  4 87  2  0 1B8.1  5 86  2  3

Example 20

GPR49 mAbs Inhibit Growth of Cancer Stem Cells In Vitro

GPR49 mAbs were tested for their ability to inhibit growth of cancerstem cells in a colon cancer stem cell tumorsphere assay.

Methods: Tumorspheres were established as previously described inExample 1, and seeded in the presence of GPR49 mAbs, or control isotypeIgG1 at 1-2000 cells per well in ultra-low-attachment plates. In oneexperiment, test and control mAbs were used to treat CT1 primary colontumorspheres that were plated at 1, 2 or 10 cells per 96 w/plate incancer stem cell medium (FIGS. 12A and 12B). In addition, a secondscreen was performed as above but with colon tumorspheres from primarycolon tumor cells CT3. CT1 and CT3 are colon tumor cell lines thatoriginated from fresh colon tumor patient biopsy samples, and were usedto establish colon cancer stem cell (tumorsphere) lines. They areconsidered “primary” lines because they are low-passage (<10 passages)derived recently from the patient primary samples. They are consideredcancer stem cell lines because they are grown under established cancerstem cell conditions (serum-free medium) and low-attachment plates(cells grow in suspension).

Results: 76C12 caused an average of 44% growth inhibition (n>10experiments) and 78F05 caused on average of 37% growth inhibition (4experiments) in the number of tumorspheres or cancer stem cells (derivedfrom CT1 colon tumor line) after 14 days (FIG. 13). Murine antibodieswere also assayed with the tumorsphere assay and resulted in 9 withgreater than 20% inhibition of cancer stem cell growth. One human mAb(76B04) and 38 murine anti-GPR49 mAbs showed no inhibition oftumorsphere growth. Data is summarized in Table 10. The top five GPR49mAbs from above were also tested in a CT3 cancer stem cell (tumorsphere)assay and showed between 52 to 37% growth inhibition (Table 11).Finally, GPR49 mAb 76C12 showed a 27% growth inhibition against cancerstem cells derived from colon tumor cell line DLD1 (average of 5experiments).

TABLE 10 CT1 Cancer Stem Cell Assay (Tumorsphere) mAb summary CT1 colonData reproducible tumorsphere in (N) mAb inhibition (%) Antibody type Pvalue experiments 76C12 44 Human p = 0.007 >10 18G7.1 37 Mouse 2G p =0.008 2 78F05 27 Human p = 0.04 4 5D6.3 30 Mouse 1st Gen p = 0.008 21B3.5 28 Mouse 1st Gen p = 0.008 3 14A8.1 27 Mouse 2G p = 0.02 2 5B10.124 Mouse 2G p = 0.008 3 14F7.1 20 Mouse 2G p = 0.002 3 5F2.5 21 Mouse1st Gen p = 0.04 1 7C3.4 20 Mouse 1st Gen p = 0.02 1 8E9.1 20 Mouse 2G

TABLE 11 CT3 Cancer Stem Cell Assay (Tumorsphere) mAb summary CT3 colontumorsphere Data reproducible mAb inhibition (%) Antibody type in (N)experiments 76C12 52 Human 3 18G7.1 42 Mouse 2G 3 78F05 37 Human 3 5D6.337 Mouse 1st Gen 3 1B3.5 46 Mouse 1st Gen 3

Example 21

In Vivo Inhibition of Primary Colon Cancer Tumors Having K-Ras, PI3K,PTEN, and p53 Mutations Using GPR49 Antibodies

We performed an experiment to determine if GPR49 antibodies wereeffective in vivo to inhibit colon cancers that had particular mutationsthat can lead to resistance to known cancer therapies. For example,patients with the KRAS mutation may have a poor response to panitumumab(Vectibix®) and cetuximab (Erbitux®) therapy in colorectal cancer. Thus,we wanted to determine if the GPR49 antibodies could provide a usefultreatment for colon cancers with these known therapeutic resistancemarkers.

Methods:

Single agent in vivo efficacy of murine antibodies 14F7.1, 18G7.1,5B10.1, 14A8.1, 1B3.5 was evaluated in a primary colon tumor xenograftmodel system using CT1 primary xenograft cells. CT1 is a primarycolorectal cancer (CRC) in vivo xenograft tumor established from a freshCRC patient tumor sample. The mutational status of the tumor cells wasdetermined by Ion Torrent® (Life Technologies, Carlsbad, Calif.) deepsequencing to identify oncogene mutations. Approximately 5-15% of CT1tumors express GPR49. SCID beige female mice were inoculated with CT1tumor cells and monitored for tumor growth. Mice were randomized intogroups of 10 when mean tumor volume reached 175 mm³. The antibodies wereadministered intraperitoneally (i.p.) at 15 mg/kg, twice weekly for 4weeks. An isotype matched antibody IgG1 was administered as a negativecontrol at 15 mg/kg twice per week for 4 weeks. Tumor volumes and bodyweights were measured 2× per week.

Results:

The antibodies inhibited tumor growth up to 40% compared to control(FIGS. 14 and 15). Antibodies 14F7 and 18G7 exhibited the highest tumorgrowth inhibition (40% and 34% tumor inhibition, respectively). The 34%and 40% tumor growth inhibition by the antibodies is an “outsized”effect since GPR49 is only expressed on approximately 5-15% of the tumorcells. This outsized effect suggests that antibody inhibition of GPR49positive cancer stem cells is targeting the source of the proliferatingtumor cells in vivo.

Example 22

In Vivo Inhibition of Primary Colon Tumors Having K-Ras, PI3K, PTEN, andp53 Mutations by GPR49 Antibodies in Combination with theChemotherapeutic Irinotecan

We also performed an experiment to determine if GPR49 antibodies incombination with well-known cancer therapeutic agents would be effectivein vivo to inhibit colon cancers that had particular mutations that canlead to resistance to known cancer therapies.

Methods:

Single agent in vivo efficacy of murine antibodies 14F7.1, 18G7.1,5B10.1, 14A8.1, 1B3.5 was evaluated in a primary colon tumor xenograftmodel system using CT1 primary xenograft cells. CT1 is a primarycolorectal (CRC) in vivo xenograft tumor established from a fresh CRCpatient tumor sample. The mutational status was determined by IonTorrent deep sequencing to identify oncogene mutations. Approximately5-15% of CT1 tumors express GPR49. SCID beige female mice wereinoculated with CT1 tumor cells and monitored for tumor growth. Micewere randomized at day 0 into groups of 10 when mean tumor volumereached 175 mm³. Chemotherapeutic Irinotecan was administered IP at 10mg/kg once per day for the first 5 days. The antibodies wereadministered intraperitoneally (i.p.) at 15 mg/kg administered twiceweekly for 4 weeks. Tumors were measured at the indicated intervalspost-inoculation for total tumor volume.

Results:

Anti-GPR49 antibodies in combination with irinotecan inhibited tumorgrowth by 57% to 65%, compared to 45% tumor inhibition by Irinotecanalone (FIG. 14, FIG. 16). GPR49 antibodies therefore enhanced theanti-tumor activity of Irinotecan by 27% to 44% in colon cancers knownto be resistant to certain available therapeutics.

Example 23

In Vivo Inhibition of Primary Colon Cancer Tumors Having K-Ras, PI3K,PTEN, H-Ras, APC, TP53, FGFR2, VANGL2, STK11, JAK2, and RB1 Mutations

We performed an experiment to determine whether GPR49 antibodies wouldbe effective as single agent inhibitors of colon cancer tumors that hadparticular mutations that can lead to resistance to known cancertherapies. We had determined, above, that GPR49 antibodies incombination with well-known cancer therapeutic agents were effective. Inthis experiment we determined the relative inhibition of tumor growthupon treatment with GPR49 antibodies compared to control treatment.

Methods:

Single agent in vivo efficacy of murine antibodies 18G7.1 and 7C3.4 wasevaluated in a primary colon tumor xenograft model system using CT3(primary colon cancer) cells. CT3 is a primary colon tumor xenograftderived from a fresh CRC patient tumor sample, and maintained in vivo atlow passage number (p<4). The mutational status was determined by IonTorrent deep sequencing to identify oncogene mutations. Approximately15-20% of CT3 tumors express GPR49. CB17-Scid female mice wereinoculated with CT3 cells and monitored for tumor growth. Mice wererandomized into groups of 10 when mean tumor volume reached 130 mm³ (Day0). Antibodies were then dosed intraperitoneally (i.p.) at 15 mg/kgtwice weekly for 4 weeks. An isotype matched antibody IgG1 wasadministered as a negative control at 15 mg/kg twice per week for 4weeks. Tumor volume and body weight was measured twice per week untilstudy completion.

Results:

The antibodies inhibited tumor growth up to 43%, compared to controltreatment (FIG. 17). This >40% tumor growth inhibition is an “outsized”effect given the GPR49 antibodies only bind to a 15-20% subpopulation ofthe CT3 tumor cells. This outsized effect suggests that inhibition ofGPR49 positive cancer stem cells is targeting the source of theproliferating tumor cells in vivo.

Example 24

GPR49 mAb Treatment Reduces Cancer Stem Cell Frequency In Vivo inPrimary Colon Tumors Having K-Ras, PI3K, PTEN, and p53 Mutations

We performed an experiment to assay for the impact of GPR49 antibodieson cancer stem cell frequency in cells derived from colon cancer tumorsthat had particular mutations that can lead to resistance to knowncancer therapies. We had observed that GPR49 antibodies demonstrated arelative impact on tumor growth that was ‘outsized’ in comparison to theproportion of tumor cells bound. This experiment was to determine theeffect of these antibodies on cancer stem cell frequency specifically.

Methods:

Isolated CT1 tumor cells from control, GPR49 mAb, Irinotecan and GPR49mAb in combination with Irinotecan treatment and control groups from thein vivo studies outlined in Example 1 and 2 were harvested, pooled,dissociated and re-implanted in a limiting dilution secondary transplantassay to measure cancer stem cell frequency. For each treatment group, 8mice were implanted with 30, 100 and 300 cells. Tumor formation (i.e.tumor take) and growth rate were monitored on a bi-weekly basis for 8weeks. To calculate the frequency of cancer stem cells in each treatmentgroup, linear regression analysis was performed with Prism GraphPad™ tocalculate the frequency of cancer stem cells in each treatment group.This assay is considered the gold-standard for measuring CSCs, becauseit is a functional CSC assay that measures the frequency of CSC clonesin any given tumor that can give rise to a new tumor in a secondaryhost. In this assay, tumors containing the CSCs to be measured wereserially transplanted into secondary recipients in limiting dilutionassay. The assay is thus a functional in vivo measurement ofself-renewal capacity, a key component of any stem cell. It does notrely on the as yet incomplete understanding and characterization of cellsurface markers or enzyme assays that are often used to measure CSCs invitro.

Results:

Tumor re-growth from GPR49 mAb and GPR49 mAb in combination withIrinotecan pre-treated tumors was significantly inhibited (FIG. 18).Greater than 62% (5/8) of mice implanted with GPR49 mAb or GPR49mAb+Irinotecan pre-treated tumors failed to show tumor formation (i.e.were tumor-free) 8 weeks post-transplant. In contrast, only 1/8 mice(13%) implanted with control treated tumors remained tumor-free 8 weekspost-transplant. Linear regression analysis showed a 3 fold reduction inthe number of CSCs after GPR49 antibody treatment compared to controls(FIG. 18).

Example 25

GPR49 mAb Treatment Reduces Cancer Stem Cell Frequency In Vivo inPrimary Colon Tumors Having K-Ras, PI3K, PTEN, H-Ras, APC, TP53, FGFR2,VANGL2, STK11, JAK2, and RB1 Mutations

We performed an experiment to determine if GPR49 antibodies had asimilar impact on cancer cells harboring a larger array of mutationsthat lead to resistances to known cancer therapies. Expanding on theresults above, we determined the effect of these antibodies on cancerstem cell frequency in cancer cell lines harboring a larger number ofmutations.

Methods:

Isolated CT3 primary tumor cells from control, GPR49 mAb, Irinotecan andGPR49 mAb+Irinotecan treated tumors from the treatment and controlgroups in the in vivo study outlined in Example 3 were harvested,pooled, dissociated and re-implanted in a limiting dilution secondarytransplant assay to measure cancer stem cell frequency. For eachtreatment group, 8 mice were implanted with 10, 30, 100 cells. Tumorformation and growth rate were monitored on a bi-weekly basis for 12weeks. To calculate the frequency of cancer stem cells in each treatmentgroup, linear regression analysis was performed with Prism Graph Pad tocalculate the frequency of cancer stem cells in each treatment group.This assay is considered the gold-standard for measuring CSCs, becauseit is a functional CSC assay that measures the frequency of CSC clonesin any given tumor that can give rise to a new tumor in a secondaryhost. In this assay, tumors containing the CSCs to be measured wereserially transplanted into secondary recipients in limiting dilutionassay. The assay is thus a functional in vivo measurement ofself-renewal capacity, a key component of any stem cell. It does notrely on the as yet incomplete understanding and characterization of cellsurface markers or enzyme assays that are often used to measure CSCs invitro.

Results:

GPR49 mAb treatment reduced CSC frequency by 5-fold compared to control(FIG. 19).

Example 26

GPR49 mAb Treatment in Combination with the Chemotherapeutic IrinotecanPrevents Colon Tumor Cells from Forming New Tumors in SecondaryRecipients

We performed an experiment to determine whether in vivo treatment ofcolon tumor xenografts with GPR49 mAb in combination with irinotecanwould prevent the colon tumor cells from forming new tumors in secondaryrecipients. New secondary tumor formation in subjects implanted withpreviously-treated tumor cells is a proxy measurement of the frequencyof tumor stem cells in the cancer cell population. We wanted todetermine the effect of GPR49 antibodies and irinotecan in combinationon secondary tumor formation in subjects receiving colon tumor cells.

Methods:

Isolated tumor cells from control or GPR49 mAb+irinotecan treated tumorsfrom the in vivo study outlined in Example 3 were harvested, pooled,dissociated and re-implanted in a limiting dilution secondary transplantassay to measure cancer stem cell frequency. For each treatment group, 8mice were implanted with 10, 30, 100 cells. Tumor formation wasmonitored on a bi-weekly basis for 12 weeks.

Results:

After a 140 day follow-up, no tumor growth was observed from a GPR49mAb+irinotecan group (0/8 mice with tumors). In contrast, 5/8 animalsimplanted with control irinotecan treated mice formed tumors with anaverage size of 1729 mm³ (FIG. 20). This showed that treatment withGPR49 antibodies destemmed the tumors and greatly reduced the capabilityof the cancer stem cells to continue proliferating, even after treatmentwith the GPR49 antibodies was discontinued.

What is claimed:
 1. A method to measure activity of a monoclonalantibody that binds a G-Protein Coupled Receptor 49 (GPR49) polypeptideto inhibit growth of cancer stem cells, comprising: growing atumorsphere of primary colon cancer stem cells, wherein the tumorsphereis derived from a single colon tumor cell and is grown in serum-free andlow-anchorage conditions; contacting a monoclonal antibody that binds toGPR49 polypeptide with a K_(d) of less than 10×10⁻⁹ M with thetumorsphere of cancer stem cells, wherein the GPR49 polypeptidecomprises the amino acid sequence of SEQ ID NO: 1, and wherein themonoclonal antibody has activity to block cancer stem cell growth invitro and antitumor activity to human cells in vivo; and measuring theactivity of the monoclonal antibody to inhibit growth of the populationof tumorspheres.
 2. The method of claim 1, wherein the antibody hasactivity to reduce growth-competent cancer stem cell abundance in atumor formation assay in a mammalian subject.
 3. The method of claim 1,wherein the antibody has activity to inhibit growth of the tumorsphereof cancer stem cells by about 25%.
 4. The method of claim 1, wherein themonoclonal antibody has activity capable of causing internalization ofGPR49 in cells contacted with the antibody.
 5. The method of claim 1,wherein the monoclonal antibody lacks specific binding to humanleucine-rich repeat containing G-protein-coupled receptor 6 (LGR6). 6.The method of claim 1, wherein the monoclonal antibody lacks specificbinding to human leucine-rich repeat containing G-protein-coupledreceptor (LGR4).
 7. The method of claim 1, wherein the monoclonalantibody specifically binds to murine GPR49.
 8. A method to determineactivity of a monoclonal antibody that binds specifically to a G-ProteinCoupled Receptor 49 (GPR49) polypeptide to inhibit growth of cancer stemcells, comprising: growing a tumorsphere of primary colon cancer stemcells, wherein the tumorsphere is derived from a single colon tumor celland is grown in serum-free and low-anchorage conditions; contacting amonoclonal antibody that binds to a GPR49 polypeptide with thetumorsphere of cancer stem cells; and measuring the ability of themonoclonal antibody to inhibit growth of colon cancer stem cells in thetumorsphere, wherein the monoclonal antibody inhibits growth of thetumorsphere by greater than about 20% as compared to a tumorsphere notcontacted with the antibody.
 9. The method of claim 8, furthercomprising measuring the activity of the monoclonal antibody to inhibitgrowth of the tumorsphere comprises comparing the growth of thetumorsphere contacted with the monoclonal antibody to growth of acontrol tumorsphere not contacted with the monoclonal antibody.
 10. Themethod of claim 9, wherein the antibody inhibits growth of thetumorsphere contacted with the antibody by greater than 40% compared tothe control tumorsphere not contacted with the antibody.
 11. The methodof claim 8, wherein the antibody is a human antibody.
 12. The method ofclaim 8, wherein the antibody is a mouse antibody.
 13. The method ofclaim 8, wherein the antibody is an IgG class antibody.
 14. The methodof claim 13, wherein the antibody is an IgG1 class antibody.
 15. Themethod of claim 8, wherein the single colon tumor cell comprises amutation in a human gene represented by a gene symbol selected from thegroup consisting of K-Ras, PI3K, PTEN, p53, H-Ras, APC, TP53, FGFR2,VANGL2, STK11, JAK2, and RB1.